Advances in Optical Physics: Volume 5 Advances in Molecular Biophotonics 9783110304596, 9783110304381

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Yong Deng, Zhenli Huang, Yu Li, Da Xing, Zhihong Zhang Advances in Molecular Biophotonics

Advances in Optical Physics

Editor‐in‐Chief Jie Zhang

Volume 5

Yong Deng, Zhenli Huang, Yu Li, Da Xing, Zhihong Zhang

Advances in Molecular Biophotonics Edited by Qingming Luo

Physics and Astronomy Classification Scheme 2010 78.20.Pa, 79.20.Ws, 87.15.−v, 87.64.kv, 87.64.M− Editor Prof. Qingming Luo Huazhong University of Science and Technology Luoyu Road 1037 430074, Hongshan District Wuhan China

ISBN 978-3-11-030438-1 e-ISBN (PDF) 978-3-11-030459-6 e-ISBN (EPUB) 978-3-11-038803-9 Set-ISBN 978-3-11-030460-2 Library of Congress Cataloging-in-Publication Data A CIP catalog record for this book has been applied for at the Library of Congress. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2017 Shanghai Jiao Tong University Press and Walter de Gruyter GmbH, Berlin/Boston Typesetting: Konvertus, Haarlem Printing and binding: CPI books GmbH, Leck Cover image: Ellende/iStock/Thinkstock ♾ Printed on acid-free paper Printed in Germany www.degruyter.com

The series: Advances in Optical Physics Professor Jie Zhang, Editor-in-chief, works on laserplasma physics and has made significant contributions to development of soft X-ray lasers, generation and propagation of hot electrons in laser-plasmas in connection with inertial confinement fusion (ICF), and reproduction of some extreme astrophysical processes with laser-plasmas. By clever design to enhance pumping efficiency, he and his collaborators first demonstrated saturation of soft-X-ray laser output at wavelengths close to the water window. He discovered through theory and experiments that highly directional, controllable, fast electron beams can be generated from intense laser plasmas. Understanding of how fast electrons are generated and propagated in laser plasmas and how the resulting electron beams emit from a target surface and carry away laser excitation energy is critical for understanding of the fast-ignition process in ICF. Zhang is one of the pioneers on simulating astrophysical processes by laser-plasmas in labs. He and his collaborators used high-energy laser pulses to successfully create conditions resembling the vicinity of the black hole and model the loop-top X-ray source and reconnection overflow in solar flares. Because of his academic achievements and professional services, Professor Zhang received Honorary Doctors of Science from City University of Hong Kong (2009), Queen’s University of Belfast (2010), University of Montreal (2011) and University of Rochester (2013). He was elected member of CAS in 2003, member of German Academy of Sciences Leopoldina in 2007, fellow of the Third World Academy of Sciences (TWAS) in 2008, foreign member of Royal Academy of Engineering (FREng) of the UK in 2011 and foreign Associate of US National Academy of Sciences (NAS) in 2012. He is the President of Shanghai Jiao Tong University, and also a strong advocate and practitioner of higher education in China.

DOI 10.1515/9783110304596-201

Preface After a three years’ effort by many top-tier scientists, the book series Advances in Optical Physics (English version) is completed. Optical physics is one of the most active fields in modern physics. Ever since lasers were invented, optics has permeated into many research fields. Profound changes have taken place in optical physics, which have expanded tremendously from the traditional optics and spectroscopy to many new branches and interdisciplinary fields overlapping with various classical disciplines. They have further given rise to many new cutting-edge technologies: – For example, nonlinear optics itself is an interdisciplinary field, which has been developing since the advent of lasers and it is significantly influenced by various technological advances, including laser technology, spectroscopic technology, material fabrication and structural analysis. – With the rapid development of ultra-short intense lasers in the past 20 years, high field laser physics has rapidly developed into a new frontier in optical physics. It contains not only rich nonlinear physics under extreme conditions, but also has the potential of many advanced applications. – Nanophotonics, which combines photonics and contemporary nanotechnology, studies the mechanisms of light interactions with matter at the nanoscale. It enjoys important applications such as in information transmission and processing, solar energy, and biomedical sciences. – Condensed matter optics is another new interdisciplinary field, which is formed due to the intersection of condensed matter physics and optics. Here, on the one hand, lasers are used as probes to study the structures and dynamics of condensed matter. On the other hand, discoveries from condensed matter optics research can be applied to produce new light sources, detectors, and a variety of other useful devices. In the last 20 years, with the increasing investment in research and development in China, the scientific achievements by Chinese scientists also become increasingly important. These are reflected by the greatly increased number of research papers pub- lished by Chinese scientists in prestigious scientific journals. However, there are relatively few books for a broad audience – such as graduate students and scholars – devoted to this progress at the frontiers of optical physics. In order to change this situation, three years ago, Shanghai Jiao Tong University Press discussed with me and initiated the idea to invite top-tier scientists to write the series of “Advances in Optical Physics”. Our initial plan was to write a series of introductory books on recent progresses in optical physics for graduate students and scholars. It was later expanded into its current form. The first batch of the series includes eight volumes: DOI 10.1515/9783110304596-202

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Advances in High Field Laser Physics Advances in Precision Laser Spectroscopy Advances in Nonlinear Optics Advances in Nanophotonics Advances in Quantum Optics Advances in Ultrafast Optics Advances in Condensed Matter Optics Advances in Molecular Biophotonics

Each volume covers a number of topics in the respective field. As the editor-in-chief of the series, I sincerely hope that this series is a forum for Chinese scientists to introduce their research advances and achievements. Meanwhile, I wish these books are useful for students and scholars who are interested in optical physics in general, one of these particular fields, or a research area related to them. To ensure these books could reflect the rapid advances of optical physics research in China, we have invited many leading researchers from different fields of optical physics to join the editorial board. It is my great pleasure that many top tier researchers at forefronts of optical physics accepted my invitation and made their contributions in the last three years. Almost at the same time, De Gruyter learned about our initiative and expressed their interest in introducing these books written by Chinese scientists to the rest of world. After discussion, De Gruyter and Shanghai Jiao Tong University Press reached the agreement in co-publishing the English version of the series. At this moment, on behalf of all authors of these books, I would like to express our appreciation to these two publishing houses for their professional services and supports to sciences and scientists. Especially, I would like to thank Mr. Jianmin Han and his team for their great contribution to the publication of this book series. At the end of this preface, I must admit that optical physics itself is a rapidly expanding forefront of science. Due to the nature of the subject area, this series can never cover all aspects of optical physics. However, what we can do – together with all authors of these books – is to try to pick up the most beautiful “waves” from the vast science ocean to form this series. By publishing this series, it is my cherished hope to attract minds of younger generation into the great hall of optical physics research. Professor Jie Zhang Editor-in-chief

Contents Preface 

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Zhihong Zhang, Qingming Luo 1 Fluorescent Protein Labeling Techniques   1 1.1 Introduction   1 1.2 Fluorescent proteins and their mutants   2 1.2.1 Colorful fluorescent proteins   3 1.2.2 Fluorescent proteins with LSSs   6 1.2.3 Photon-activatable and photon-switchable fluorescent proteins   7 1.2.4 Light-sensitive fluorescent proteins   9 1.2.5 Timer fluorescent protein   10 1.3 Reporter fluorescent protein probes   11 1.3.1 Tracking proteins in live cells   11 1.3.2 Monitoring of gene expression in live cells   14 1.3.3 Biological applications of photon-switchable proteins and photon-activatable proteins   15 1.4 Functional fluorescent protein probes   18 1.4.1 Redox probes   18 1.4.2 ATP fluorescent protein probes   21 1.4.3 pH probes   22 1.4.4 Voltage-sensitive probes   24 1.4.5 Calcium probes   26 1.4.6 Mercury ion probes   30 1.4.7 Copper ion probes   30 1.4.8 Zinc ion probes   31 1.5 Fluorescence resonance energy transfer (FRET) probes   32 1.5.1 Introduction of FRET   32 1.5.2 FRET imaging in cell biology research   34 1.5.3 Intramolecular FRET probes   36 1.5.4 Intermolecular FRET probes   40 1.6 BiFC technology based on fluorescent proteins   43 1.6.1 Establishment of the BiFC detection method   43 1.6.2 Characteristics of BiFC technology   44 1.6.3 Applications of BiFC technology   46 1.6.4 Quantitative detection of protein interaction based on fluorescence signal of BiFC   48 1.6.5 Limiting factors of bimolecular fluorescent complementation   48 1.6.6 Outlook for BiFC   49 1.7 Intravital applications of fluorescent proteins in tumor imaging   49

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1.7.1 In vivo tumor optical imaging based on endogenously expressed fluorescent protein   50 1.7.2 Optical imaging of tumor in vivo with targeting FP probes   59 1.7.3 Prospects   62 1.8 Applications of fluorescent protein transgenic mice in intravital immune optical imaging   63 1.8.1 Fluorescent protein transgenic animal models   63 1.8.2 Applications of fluorescent protein–labeled pathogens in infection and immune imaging   68 Bibliography   74 Yu Li, Lingyu Zeng, Zhihong Liu, Jingui Qin 2 Two-photon Molecular Probe   93 2.1 Introduction of two-photon absorption   93 2.1.1 The basic concept of 2PA   93 2.1.2 Measurements of 2PA effect   96 2.1.3 Introduction to application of 2PA effect   99 2.2 Molecular design and structure–property ­relationships of organic TPA materials   103 2.2.1 One-dimensional asymmetric D–π–A molecules   104 2.2.2 One-dimensional symmetric molecules   107 2.2.3 Porphyrins and expanded porphyrinoids   112 2.2.4 Multidimensional branched 2PA materials   116 2.3 The development of two-photon fluorescent probes   119 2.3.1 Brief introduction to response principle of fluorescent probes   120 2.3.2 Traditional fluorescent probes for two-photon imaging   122 2.3.3 Typical fluorophores for TP probes   122 2.3.4 Research development of TP probes   125 2.3.5 Research prospection of TP probes   181 Acknowledgment   186 Bibliography   186 Zhenli Huang, Yina Wang, Fan Long, Zhe Hu, Zeyu Zhao 3 Super-resolution Localization Microscopy   194 3.1 Introduction and background   194 3.1.2 Resolution limit of optical microscope   196 3.1.3 Improving the resolution of optical microscope   197 3.1.4 A historical overview of super-resolution localization microscopy   197 3.1.5 Breaking the resolution limit by single-molecule localization   199 3.2 Fluorescence probes for super-resolution ­localization microscopy   200 3.2.1 Ensemble and single-molecule fluorescence  200

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Fluorescence probes and specific labeling  3.2.2  202 3.2.3 Fluorescence ON/OFF control   204 3.2.4 Choosing the right fluorescence probes   205 3.3 Methods and instrumentation in super-­resolution localization microscopy   208 3.3.1 Super-resolution localization microscopy methods: PALM versus STORM   208 3.3.2 Super-resolution localization microscopy methods: Others   210 3.3.3 Instrumentation in super-resolution localization microscopy: Basic structure   210 3.3.4 Instrumentation in super-resolution localization microscopy: Key components   211 3.3.5 Instrumentation in super-resolution localization microscopy: A typical setup   214 3.3.6 Advances in super-resolution localization microscopy: Multicolor and 3D imaging   216 3.3.7 Commercial super-resolution localization microscopes   217 3.4 Data analysis in super-resolution localization microscopy   218 3.4.1 Theoretical localization precision   218 3.4.2 Practical aspects for determining spatial resolution   219 3.4.3 Single-molecule localization for sparse emitters   220 3.4.4 Single-molecule localization for high-density emitters   223 3.4.5 Key steps in image analysis and reconstruction   225 3.4.6 Data analysis software   226 3.5 Example applications in super-resolution ­localization microscopy   227 3.5.1 Imaging in 2D   227 3.5.2 Imaging in 3D   228 3.6 Conclusions and future prospects   229 Bibliography   230 Da Xing, Sihua Yang 4 Photoacoustic Molecular (Functional) Imaging   235 4.1 Introduction   235 4.2 PAI principle, algorithm, and system   238 4.2.1 PAI principle   238 4.2.2 Excitation of photoacoustic signal   239 4.2.3 Photoacoustic scanning method and its imaging algorithm  4.2.4 PAI system   254 4.2.5 Special problems involved   270 4.3 Domestic and foreign statuses   275 4.3.1 Foreign research status   275 4.3.2 Domestic research status   286

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4.4 Application development trend   298 4.4.1 Application research of photoacoustic microcirculation imaging and early tumor detection and treatment monitoring   299 4.4.2 Research on application of living body photoacoustic blood function parameters (blood oxygen and carbon oxygen saturation) detection   306 4.4.3 Application research on photoacoustic identification and imaging of vulnerable plaque components in blood vessels   309 4.4.4 Application research on thermoacoustic imaging in testing of lowdentistry foreign bodies   313 4.4.5 Application research on thermoacoustic imaging in testing of breast cancer   315 Bibliography   318 Yong Deng, Xiaoquan Yang, Qingming Luo Optical Molecular Imaging for Small Animals in vivo  5  324 5.1 Models of light propagation in tissue   324 5.1.1 Introduction   324 5.1.2 Light transport equation   325 5.1.3 Diffusion approximation method   329 5.1.4 Monte Carlo method   333 5.2 Diffuse optical tomography   343 5.2.1 Introduction   343 5.2.2 DOT mode   344 5.2.3 Image reconstruction methods in DOT   348 5.2.4 Applications in biomedical research   352 5.3 In vivo optical molecular imaging of small animals   354 5.3.1 Introduction   354 5.3.2 Planar fluorescence molecular imaging   355 5.3.3 Fluorescence molecular tomography   361 5.3.4 Bioluminescence tomography   370  375 5.4 Multimodality molecular imaging of small animals in vivo   375 5.4.1 Introduction  5.4.2 Multimodality molecular imaging systems   376 5.4.3 Image reconstruction and multimodal image fusion   382 5.4.4 Applications in biomedical research   387 Bibliography   393 Index 

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Zhihong Zhang, Qingming Luo

1 Fluorescent Protein Labeling Techniques 1.1 Introduction The green fluorescent proteins (GFPs) from Aequorea victoria, red fluorescent proteins (RFPs) from coral or anemone, and their mutants have been widely used as marker and reporter molecules in dynamic monitoring and visualization of important molecular life events, including regulation of gene expression, protein spatial location and orientation, interactions among biological molecules, tumor growth and angiogenesis, and genetics as well as development and evaluation of the efficacy of new drugs and study of their mechanisms of actions. These new materials provide effective tools for research on biological molecular functions and dynamic changes in living cells and organisms. Due to the precise subcellular localization ability and good biocompatibility of genetically encoded molecular probes and because the amount of optical signal molecules is not reduced by cell division, it is possible to conduct long-term (a few days to several months) dynamic monitoring of cellular fates and molecular events in living cells and organisms. The 2008 Nobel Prize in Chemistry was awarded to Martin Chalfie, Osamu Shimomura, and Roger Y. Tisen for their outstanding contribution to the discovery and biological applications of GFPs. The discovery of novel fluorescent proteins, generation of their mutants, and the subsequent development and optimization of functional fluorescent protein probes have greatly promoted the development of modern biology with wide applications in many areas of biological research. In combination with functional fluorescent protein-based molecular probes, optical microscopy imaging methods such as fluorescence resonance energy transfer (FRET), fluorescence recovery after photon bleaching (FRAP), fluorescence redistribution after photon activation (FRAPa), fluorescence lifetime imaging microscopy (FLIM), and bimolecular fluorescence complementation (BiFC) have launched a new chapter in dynamic studies of biomolecules in living cells and exploration in many new areas of knowledge in biology. Among these methods, FRET, which operates on the “optical scale” of the 1.0- to 10.0-nm range, is the most widely used technology in the study of protein function. Molecular probes designed based on FRET principles are used for dynamic monitoring of the distance between protein – protein interactions, protease activation, protein conformational changes, etc. and has become a routine tool in the study of protein molecular events during signal transduction in living cells. In research on optical imaging of tumors in vivo, using GFP, RFP, and their mutants as tumor markers [1], scientists have demonstrated

Zhihong Zhang, Qingming Luo: Huazhong University of Science and Technology, Email: qluo@mail. hust.edu.cn; [email protected]. DOI 10.1515/9783110304596-001

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long-term dynamic optical visualization of tumor growth, metastasis, and treatment in small animals [2], thus providing great and valuable means for studying tumor molecular mechanisms and screening and evaluation of new drugs. To examine immune response in vivo, using GFP transgenic mice and the two-photon microscopic imaging technique, the immune response mechanism underlying neutrophil resistance to pathogens [3] and the motility of fluorescent protein-labeled immune cells during infection were dynamically observed and characterized, thus enhancing understanding of how the immune system participates in the etiology, progression, and cure of diseases [4]. However, the use of fluorescent proteins is limited in deep tissue imaging due to the opaque characteristics of biological tissue and interference from hemoglobin. Fortunately, new far-red fluorescent proteins (far-RFPs) and near-infrared fluorescent proteins (near-IFP), such  as mKate (λex/em: 588/635 nm) [5], mNeptune (λex/em: 600/650 nm) [6], and iRFP (λex/em: 701/719 nm) [7, 8], have greatly improved the acquisition ability of depth information acquired via in vivo optical imaging. To further enhance the brightness of fluorescent proteins, optimization of their physicochemical parameters and development of far-RFP or near-IFPs with more abundant spectral features are important directions for future application of fluorescent proteins to in vivo optical imaging. Similarly, another hot research area is development of fluorescent protein probes with large Stokes shifts (LSSs) that are well suited to FRET imaging and also photon-activatable/photon-switchable fluorescent proteins with better performance that are more appropriate for super-resolution imaging. Together with continuous advances in optoelectronic information technology, optical molecular imaging technologies based on fluorescent proteins are focused on simultaneous and dynamic visualization of multiple molecular events at different levels, such as cells, cell networks, tissues, organs, and individuals, thus providing an indispensable research tool for revealing the nature of biological activities and early diagnosis and treatment of major diseases. This chapter introduces fluorescent protein markers and their biological applications in the following seven categories: (1) fluorescent proteins and their mutants, (2) fluorescent protein reporter, (3) functional fluorescent protein probes, (4) FRET probes, (5) BiFC technology based on fluorescent proteins, (6) applications of fluorescent protein markers to in vivo optical imaging of tumors, and (7) use of fluorescent protein transgenic mouse models for in vivo immune optical imaging.

1.2 Fluorescent proteins and their mutants Since fluorescent protein was first found and used in biological research, scientists have obtained a variety of new fluorescent proteins from organisms and have constantly optimized such physicochemical properties as fluorescence brightness,



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stability, acid resistance, and spectral characteristics, among others. This section introduces the colorful fluorescent proteins, large Stokes-shifted fluorescent proteins, photon-activatable and photon-switchable fluorescent proteins, light-sensitive fluorescent proteins, and timer fluorescent proteins.

1.2.1 Colorful fluorescent proteins 1.2.1.1 History of the discovery of fluorescent proteins Shimomura Osamu isolated and purified the wild-type GFP (wtGFP) from the jellyfish A. victoria and another luminescent protein aequorin for the first time during the period 1960–1970 and studied the luminescence principle and characteristics of GFP. In the jellyfish A. victoria, aequorin emits blue light when it binds with Ca2+, and a portion of the blue light energy is transferred to GFP to emit light, which shifts the emission spectrum towards green [9]. However, molecular biologists did not pay attention to GFP until 1992 when Prasher et al. [10] successfully cloned the wtGFP gene. Unfortunately, Prasher did not continue his work due to lack of research funding, but he presented GFP cDNA to other laboratories. In 1994, Chalfie [11] knocked out several upstream amino acid residues of GFP and first successfully expressed GFP in Escherichia coli and nematodes, thus demonstrating use in cross-species. Nearly simultaneously, the Tsuji Frederick lab reported the results of GFP fusion expression a month later [12]. Similar to wtGFP, this GFP is able to fold at room temperature and emit fluorescence when it is excited by light, without the involvement of other molecules, as in jellyfish. However, despite emitting fluorescence independently, this GFP has a number of disadvantages. It has two excitation peaks; is sensitive to pH and chloride; and has low quantum yield, low light stability, and protein folding ability at 37°C. The crystal structure of the S65T GFP mutant was first reported in 1996 by the Remington team [13].A month later, the Phillips lab also independently completed a structural analysis of wtGFP [14]. The study of the crystal structure provided an important theoretical basis for the analysis of formation of fluorophores and interactions between the adjacent amino acid residues. Since then, many research groups have produced various GFP mutants by changing these residues directly or through random mutations. Roger Y. Tsien studied the chemical mechanism of GFP luminescence and obtained a series of fluorescent protein mutants with different colors. Subsequently, scientists have continuously explored and developed fluorescent proteins that are more stable and have more prominent spectral characteristics and also developed functional fluorescent probes to meet research needs using existing fluorescent proteins. The 2008 Nobel Prize in Chemistry was awarded jointly to Martin Chalfie, Osamu Shimomura, and Roger Y. Tisen, for the discovery and development of the green fluorescent protein, GFP.

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1.2.1.2 Structure of GFP wtGFP consists of 238 amino acids, has a molecular weight of 27 kD, and absorbs blue light with two major peaks (major peak at 395 nm and minor absorption peak at 470 nm). The structure of wtGFP shows a typical cylindrical structure with a hydrophobic center consisting of 11 beta sheets. The cylindrical structure, with a diameter of approximately 2.4 nm and a height of approximately 4.2 nm, is highly dense and stable under normal conditions, which ensures stable optical properties. The central fluorophore of the wtGFP is a covalent structure composed of a beta sheet and an alpha helix, that is, 4-(p-hydroxybenzylidene) imidazolidin-5-one, HBI, and HBI in misfolded GFP does not display fluorescence. A chromophore is formed when HBI interacts with the side chains of the cylindrical structure to induce Ser65-Tyr66-Gly67 tripeptide cyclization and convert HBI to the phenol salt form. Mutation of the side chain affects this post-translational modification process. Therefore, different amino acid side chains will change the fluorescence spectra, strength, and stability of the fluorophore by affecting the formation of hydrogen bonds and electron clouds. The dense cylindrical structure of the fluorescent protein prevents the fluorophore from interacting with polar molecules, thereby protecting the fluorophores from quenching by water molecules [15].

1.2.1.3 The GFP family After he revealed the luminescence mechanism of GFP, Roger Y. Tsien obtained a series of fluorescent mutants with different colors. The chromophore is formed by residues 65–67, which are Ser-Tyr-Gly in the native protein. Ser65 is a common mutation site. Three mutants of GFP with different colors, that is, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) were first obtained through mutations of the chromophore in combination with alterations of other specific sites. To further optimize the physicochemical properties of fluorescent protein, additional GFP mutants were produced using genetic engineering methods. For example, the Ser65→Thr mutant (S65T) of wtGFP shows greatly increased brightness and photon stability and had the longest wavelengths of excitation and emission (488 and 509 nm), which closely resembled those of fluorescein isothiocyanate isomer (FITC), a wtGFP suitable for systematic uses in optical imaging [16]. In 1995, the F64 L mutant, that is, enhanced GFP (EGFP) was introduced, and its better folding efficiency at 37°C made it more suitable for mammalian cells. This superfolder GFP produced through a series of mutations was able to fold and mature more rapidly [16]. Many other mutants with different colors have been subsequently developed via gene mutations: BFPs (EBFP, EBFP2, Azurite, mKalamal), CFPs (ECFP, Cerulean, CyPet), and YFPs (YFP, Citrine, Venus, YPet). The main reason for the red shift of YFP is that the T203Y mutation alters the interaction between lysine residues and



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the chromophore and thus changes the π electrons. Currently, the mutants of CFP and YFP are widely used in FRET experiments, and probes aimed at such targets as calcium ions, glutamic acid, and protein phosphorylation have been developed [17, 18]. Additional information on FRET is introduced in detail in Section 1.5.

1.2.1.4 The orange and red fluorescent protein series DsRed is the first known RFP obtained, and additional excellent mutants were produced based on DsRed, including mBanana (λex/em: 540/553 nm), mOrange (λex/em: 548/562 nm), dTomato (λex/em: 554/581 nm), mTangerine (λex/em: 568/585 nm), mStrawberry (λex/em: 574/596 nm), and mCherry (λex/em: 587/610 nm) [19]. Certain differences exist between the spectral characteristics and physicochemical properties of these fluorescent proteins, which are named after fruit. By virtue of rapid maturation, the monomeric fluorescent protein mCherry has become the most widely used choice among RFPs. Karasawa et al. [20] developed a monomer orange fluorescent protein mKO (λex/em: 548/561 nm) with excellent performance using point mutation of Kusabira Orange (an orange fluorescent protein) from Fugia concinna. Hidekazu, etc. obtained mKOκ by screening a large number of mutants of mKO generated by random mutagenesis. The main advantages of mKOκ are faster maturation and higher brightness [21]. TagRFP (λex/em: 555/584 nm) is a monomeric RFP from anemone (Entacmaea quadricolor) with the currently known maximum brightness [22]. mRuby is another monomer fluorescent protein obtained by mutation of TagRFP on eqFP611, which has high homology with TagRFP. mRuby is an excellent FRET receptor because its extinction coefficient reaches 112,000 M−1cm−1, which is much higher than that of TagRFP. However, the quantum yield of mRuby is slightly lower than that of TagRFP [23]. In addition, the C-terminal peroxisomal-targeting sequence (Ser-Lys-Leu, SKL) of the matrix has been removed in mRuby, thus avoiding specific localization of the protein in peroxisomes. The mOrange2 and TagRFP2 proteins obtained by Shaner et al. [19, 24] via screening are more suitable for long-term continuous imaging due to their especially strong photon stability (increased by a factor of 25 and 9, respectively, compared with mOrange and TagRFP).

1.2.1.5 Far-IFPs and near-IFPs The near-infrared band (650–900 nm) is considered the “optical window” of biological tissue imaging, and therefore red-shift far-red or near-IFPs aid in increasing the in vivo imaging depth and enhancing the signal-to-noise ratio (SNR). A point mutant of TagRFP known as mKate (λex/em: 588/635 nm) is a new type of monomer far-red fluorescent protein, and its dimer is Katushka [5]. Related research in transgenic Xenopus imaging indicates that mKate and Katushka are superior to EGFP and RFP for in vivo

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animal imaging because they have better imaging depth due to their spectral characteristics. mKate2 was developed by V38A/S165A/K238R mutation from mKate. The brightness of mKate2 is doubled compared with that of mKate, and the spectral characteristics are more stable [25, 26].Additionally, TaqRFP657 (λex/em: 611/657 nm) and mNeptune (λex/em: 600/650 nm) were obtained based on mKate2, with their spectra red shifted even further. Roger Y. Tsien group reported a infrared fluorescent protein known as IFP1.4 in 2009, which was the result of screening bacterial phytochromes [27], and IFP1.4 is the first fluorescent protein that emits light with a wavelength longer than 700 nm. Because it must absorb and use biliverdin as a chromophore, IFP1.4 is not a fluorescent protein in the strict sense. Nevertheless, biliverdin widely exists as a metabolite in mammalian cells and has notably low biological toxicity. Because IFP1.4 depends on exogenous biliverdin to produce fluorescence and its brightness is weak, its application has been restricted in optical imaging of small animals. Based on this information, Filonov et al. [7] further modified bacterial phytochromes and invented iRFP in 2011. The excitation and emission spectra of iRFP are further red shifted (λex/em: 690/713 nm) than IFP1.4, and iRFP also offers increased brightness, photon stability, and SNR. Furthermore, iRFP does not require exogenous biliverdin. Inspired by this report, Auldridge et al. [28] screened bacterial phytochrome mutants to obtain Wisconsin infrared phytofluor (Wi-Phy) in 2012, and its maximum excitation and the emission wavelength were red shifted to 701 nm and 719 nm, respectively, although its brightness is slightly weaker than that of IFP1.4. In 2013, Shcherbakova and Verkhusha [29] invented iRFP670 and iRFP720 via further optimization of iRFP, thus realizing multicolor in vivo tumor imaging. Among these materials, iRFP720 is 10 % brighter than iRFP670 in HeLa cells, and its brightness is approximately 14 times that of IFP1.4. Because biologists have an increasing understanding of in vivo imaging and are utilizing it more broadly, a great need exists for fluorescent proteins with more extensively red-shifted spectra and high brightness.

1.2.2 Fluorescent proteins with LSSs The Stokes shift is the difference between the peak positions of the absorption and emission spectra of the same electronic transition. Fluorescent molecules with LSSs generally refer to molecules whose excitation and emission peaks are farther than 100 nm apart, and in multicolor imaging, LSSs can effectively reduce spectral cross talk. The use of LSS fluorescent proteins to label cells or molecules for fluorescence imaging offers several advantages: (1) Use of a single-excitation light for simultaneous imaging of multiple fluorescent molecules due to the large difference between the emission and excitation peaks of molecules with LSSs; (2) These materials can effectively avoid direct excitation of the acceptor molecules if used as a donor molecule in FRET imaging; (3) RFPs with LSSs are better for two-photon imaging because



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the two-photon excitation peak is located in the near-infrared wavelengths, and the emission wavelength is located in the red wavelengths, and thus both the excitation and emission are less absorbed by the tissues, making it possible to obtain clearer pictures for in vivo imaging [30]. There are some LSS fluorescent proteins reported currently, included T-Sapphire  [31], mAmetrine [32], mKeima [33], mLSS Kate1, and mLSS Kate2 [30]. wtGFP (λex/em: 395/509 nm) is a large Stokes shift fluorescent protein [15]. The small excitation peak at 475 nm was removed from the parent wtGFP through T203I mutation, and the obtained mutant Sapphire has a Stokes shift of 100 nm. The further developed T-Sapphire is more stable and more suitable for formation of an FRET pair together with the OFP or RFP [31]. mAmetrine (λex/em: 406/526 nm) is another LSS mutant that originates from GFP. It has been recorded that mAmetrine and td-Tomato can form an FRET pair and achieve FRET imaging of dimolecular events within a single cell in combination with mTFP/Citrine [32]. The first reported LSS-RFP (LSS RFP) is mKeima (λex/em: 440/620 nm) [33]. Certain researchers used a single 458 nm light to excite CFP and mKeima simultaneously, and developed dual-color imaging excited by a singleexcitation light source [33]. mKeima has a second excitation peak at 584 nm, and follow-up reports focused on how to remove the interference of the second excitation peak in multicolor imaging [34]. LSS mKate1 and LSS mKate2 are new monomer mutant fluorescent proteins based on mKate that have characteristics of rapid maturation and stability in an acidic environment, and their excitation and emission peaks are 463/624 nm and 460/605 nm, respectively [30]. However, these LSS red fluorescent materials (e. g., mKeima [33], mLSSKate1, and mLSS Kate2 [30]) all have significant limitations in applications, including low brightness and low thresholds for photon bleaching. Invented by Yang and Zhang [35], mBeRFP (λex/em: 446/611 nm) is the brightest LSS RFP, which originated from mKate, and its brightness is 3 times higher than that of mKeima or mLSSKate2, together with a maturation speed that is 2.2 times as fast and an anti – photo-bleaching performance that is doubled.

1.2.3 Photon-activatable and photon-switchable fluorescent proteins 1.2.3.1 Irreversible photon-activatable and photon-switchable fluorescent proteins Unlike the reversible photon-activatable fluorescent protein (PA-FP), an irreversible PA-FP can be induced from the dark state to the bright state or its spectra can shift only once, and if it were subjected to continuous stimulation, it would show fluorescence emission and photon-bleaching similar to that of an ordinary fluorescent protein, until it loses its luminosity. More numerous irreversible PA-FP types and mutants exist because the photon-activated chemical process is easier than that of the reversible PA-FP. Thus far, a number of excellent proteins have been discovered, as represented

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by PA-GFP, and they have been widely used in many areas, from ultra-resolution imaging to tracking of cells in vivo [36–40] (for additional details, see Section 1.3.3). (1) PA-FP The first reported PA-FP was produced by T203H point mutation from wtGFP. If this protein is activated at 405 nm before imaging, it can be used in continuous imaging at 488 nm. The fluorescence intensity can differ by up to 100 times before and after activation at 405 nm. Thus, the difference of fluorescence intensity between the activated region and the nonactivated region can be used to dynamically capture molecular dynamics within the target region. Based on this technique, photon-activated localization microscopy (PALM) further expands the applied range of PA-GFP. The red PA-FP known as PA-mCherry1 was subsequently developed, and dual-color super-resolution imaging of biomolecules in a single cell was achieved by combination of the different spectral properties of PA-GFP and PA-mCherry1 [40, 41]. (2) Spectral-shifted photon-switchable fluorescent proteins (PS-FPs) The emission wavelength of such fluorescent proteins changes after laser irradiation at a certain wavelength: 1. The emission peak of PS-CFP2 migrated from 468 nm to 511 nm after activation, and the fluorescent intensity increased by a factor of 1,500. It has been reported that a PS-CFP2-labeled dopamine transporter receptor could be used to study the real-time dynamic characteristics of the receptor within a specific region based on the PS-CFP2 spectral shift [42]. 2. Kaede is a cloned fluorescent protein from Lobophyllia hemprichii [43]. The emission peak migrated from 518 nm to 582 nm after activation by ultraviolet light. It is now known that the peptide chain of its chromophores breaks up after activation, thus changing the fluorescence properties of Kaede. The major limitation of Kaede is its tetramer structure, which hampers the labeling of target proteins. 3. EosFP is another PS-FP derived from coral that can be activated by a 390-nm laser, and the emission peak migrates from 516 nm to 581 nm [44]. EosFP is a monomeric fluorescent protein widely used in super-resolution imaging. In 2012, Xu et al. developed mEos3.1 and mEos3.2 by mutating the key sites of mEos, and these mutations overcame mEos’s tendency to form dimers or multimers at high concentration. The mEos3 series are PA-FPs with the best monomeric performance known so far [45]. 4. Dendra is a monomeric fluorescent protein derived from Octocoral dendronephthy in which the emission peak migrated from 505 nm to 575 nm after activation by blue light, and its spectral properties are similar to that of Kaede. After activation, the red fluorescence Dendra is enhanced by a factor of 350, and the green fluorescence is decreased by a factor of 5, thus showing a 1,400-fold difference between the background and fluorescence signals [46].



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1.2.3.2 Reversible photon-activatable or photon-switchable fluorescent proteins Reversible photon-activated fluorescent proteins (RPA-FPs) can be controlled by light with a specific wavelength, intensity, and duration and are switchable between bright and dark states. FP595 was the first reported RPA-FP, but due to its tetramer characteristic, it was soon replaced with other excellent monomeric fluorescent proteins. Dronpa (λex/em: 503/518 nm) is derived from comb coral (Coral pectiniidae), and its fluorescence is quenched after activation by light with a wavelength from 470 to 510 nm. After quenching, Dronpa in the dark state can be reactivated by a 400-nm laser and emits green fluorescence, and this quenching-activation cycle is repeatable [47]. Thus far, the excellent mutants (such as Dronpa) have tolerance to hundreds of repetitions of the “light switching” cycle. In the photon activation process, the chromophore of fluorescent proteins undergoes cis-trans isomerization and emits fluorescence in the cis-structure but no fluorescence in the trans-structure. However, slow photon switching is a major drawback of Dronpa. To increase the speed of photon switching, mutagenesis screening techniques were used to produce two Dronpa mutants of rsFastlime and Dronpa M159T, and their speed of bright-dark state conversion was increased 1,000 times compared with that of Dronpa [48]. Subsequently developed mutants of Dronpa-Padron and bsDronpa (both with a broad absorption spectrum) are more suitable for bimolecular multicolor super-resolution imaging [39]. Padron and bsDronpa have opposite photon switch features: bsDronpa can be activated by violet, and Padron is activated by blue and quenched by violet. In addition, Kindling (KFP1) from pink anemone (Anemonia sulcata), which does not emit fluorescence by itself, emits red fluorescence (600 nm) after activation by ­low-intensity laser ­(525–580 nm) irradiation. Its fluorescence disappears as the illumination ends; thus, KFP1 must be in an activated state to maintain fluorescence [38].

1.2.4 Light-sensitive fluorescent proteins Light-sensitive fluorescent proteins are a class of fluorescent proteins whose center chromophores can be inactivated by illumination and lose their fluorescence and produce toxicity at the same time, through chromophore-assisted light inactivation (CALI) [49]. KillerRed was the first of the light-sensitive fluorescent proteins found to display strong photon toxicity, and it is 1,000 times more toxic than EGFP [50]. As a mutant of a nonfluorescent red chimeric protein-anm2CP, which exists in jellyfish, KillerRed is a dimer, with its excitation and emission peaks located at 584 nm and 610 nm, respectively. Why does the light-sensitive fluorescent protein produce toxicity? The chromophores in the barrel structure are quenched via assisted light inactivation (CALI) when the fluorescent protein is illuminated, thereby producing reactive oxygen species (ROS) that are toxic to cells. Certain researchers took advantage of the utility

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of KillerRed to achieve the pointed destruction of nucleic acids via the expression of KillerRed targeting to DNA and RNA in tumor cells. If KillerRed can be expressed in the mitochondria, it can launch cell apoptosis. Research shows that the fastest and most effective way to kill cells is expression of KillerRed in the cell membrane. Other researchers used genetic engineering methods to connect the anti-HER2 antibody 4D5scFv to KillerRed to form 4D5scFv-KillerRed [51], which can target tumor cells that express HER-2 receptors and kill the tumor cells effectively. KillerRed also can be used to detect protein – protein interactions [52]. The ROS produced by excited KillerRed can inactivate not only the proteins that interact with KillerRed but also the proteins that interact with the protein that interacts with KillerRed. Therefore, KillerRed provides a PPI research technique that is different from and complementary to FRET and can be useful for distances over 10 nm. The three-dimensional structures of certain macromolecules, such as the transcription complex, ribosome, and proteosome, are larger than 10 nm. Therefore, it is difficult to apply FRET technology to PPI research on these macromolecules. The ROS released from KillerRed can reach a range from 10 nm to 50 nm, which can be useful for PPI research, as mentioned previously.

1.2.5 Timer fluorescent protein A mutant from the fluorescent protein drFP583 originated from coral is the reported first timer fluorescent protein [53]. Based on previous work, Irving Weismann transformed drFP583 using error-prone PCR to develop a mutant E5 that can change color over time from green to yellow, to orange, and finally, to red. The GFP fluorescence implies that E5 contains a fluorophore similar to GFP and can transform into a red fluorophore. Yellow and orange fluorescence imply that E5 contains both GFP and RFP fluorophores. In the research on this changing fluorescence, we found that the mutation drEP583-E5 has a slowly changing fluorescent spectrum in reducing buffer, which implies that an oxidizer might play a role in the fluorophore change process [54, 55]. Comparing the deFP583 and its E5 mutant, we found two mutant sites of V105A and S197T in E5, and the quantum yield of E5 was twice that of drFP583 but with the same spectrum. The S197T mutant can maintain the fluorophore benzene ring structure that yields change in the fluorescence spectrum. In vitro experiments showed that the transfer efficiency of the color was independent of the protein concentration, which implies that the optical property of the FPs is independent of expression level in the cell. In general, E5 can be used for dynamic monitoring of the gene expression process. A green signal means that the promoter has just been activated; a yellow-orange signal shows that the promoter is undergoing activation, and red signal indicates that the promoter has been stopped. In 2004, Konstantin et al. found that the DsRed, a member of the RFP family, has a green signal. In the maturation process, the green signal appeared earlier than the red signal in DsRed, but the green signal was reduced quickly in the maturation process. It was found later on that DsRed has a fluorophore similar to that of GFP [56].



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Based on this finding, the Konstantin group studied the relationship between the DsRed spectrum and the ripening time and explained the mechanism of color changes in its red fluorophore. These researchers also made use of this property of DsRed to infer the early expression of promoters.

1.3 Reporter fluorescent protein probes Definition of protein distribution and dynamic behavior in live cells is an important element for clarifying its functions. Direct or indirect labeling of proteins with fluorescent in combination with modern microscopic imaging technologies has become one of the most important tools for studying proteins.

1.3.1 Tracking proteins in live cells 1.3.1.1 Labeling specific proteins Live cells contain a great number of proteins, and their subcellular locations, molecular chaperones, and dynamic behaviors are directly related to the functions and life activities of the complexes in which the proteins reside. However, it is quite difficult to identify and track thousands of proteins without specific labels. Therefore, we must first label the proteins specifically and stably, so that we can subsequently study the proteins’ spatial locations, orientations, and functions. Fluorescence labeling is the most frequently used of all labeling methods. Based on their encoding characteristics and good biocompatibility, stability, and independence, fluorescent proteins have been widely used by biologists when coupled with target proteins for fusion expression, thus labeling the target proteins with fluorescent tags. In the last 10 years, tens of fluorescent proteins have been generated and applied, and their monomers and solubilities are also excellent. The fluorescent protein emission spectrums have covered the blue, cyan, green, yellow, red, far-red, and even the near-infrared bands. At the same time, many fluorescent proteins with LSSs have been reported in succession. Thus, scientists can label different proteins in a cell and perform synchronous tracing with these colorful fluorescent proteins, which can offer more enriched information on the proteins’ actions. The proteins’ spatial locations are closely associated with their functions. Different subcellular components in cells contain specific marker proteins. For example, cytochrome C oxidase could be a marker of mitochondria, and calnexin is the marker of the endoplasmic reticulum. Certain specific amino acid sequences can locate fluorescent proteins to specific organelles or components. By fusing fluorescent proteins with the marker proteins of the subcellular components or specific aminoacid targeting sequences and transforming cells with the genes of fusion proteins, we can

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directly examine the structure of subcellular components and the distribution of specific proteins using fluorescence imaging [57]. For example, fused protein (expression format: FPs-target protein-N/C terminal (relative to FPs), the length of sequence which link FPs and proteins) mCherry-H2B-N-6 is used to label chromosomes or chromatin, mWasabi-mitochondria-N-7 is used to label mitochondria, mCitrine-Cx13-N-7 is used to label gap junction structures, mCerulean-cytokeratin-N-17 labels intermediate filaments of epithelial cells, mApple-annexin-C-12 labels the distribution of phospholipid-binding proteins, mEmerald-vinculin-C-23 labels the distribution of adhesion plaques, mEGFP-EB3-N-7 labels the distribution of microtubule-associated protein EB3, mKO-Golgi-N-7 labels the Golgi apparatus, mCherry-vimentin-N-7 labels the distribution of vimentins, mTagBFP-lysosomes-C-20 labels lysosomes, mCerulean-lamin B1-C-10 labels the distribution of lamin B, mKO2-farnesyl-C-5 labels the distribution of Pennsylvania acyls, mTFP1-β-actin-C-7 labels microfilaments, mOrange2-peroxisomes-C-2 labels peroxisomes, mApple-VASP-C-5 labels the distribution of vasodilator-stimulated phosphoproteins, mEmerrald-α-tubulin-C-6 labels microtubules, mCherry-clathrin (light chain)-C-15 labels mesh protein-mediated vesicles, mEGFP-VE-cadherin-N-10 labels the distribution of vascular endothelial cadherins, TagRFP-T-endosomes-C-14 labels endosomes, mVenus-CENPB-N-22 labels centromeres, mCerulean-zyxin-N-6 labels the distribution of zyxins, mKO-fibrillarin-C-7 labels the distribution of fibrillarins, mECFP-endoplasmic reticulum-N-5 labels the endoplasmic reticulum, mApple-α-actinin-N-19 labels the distribution of actinins, mEmerrald-LC-myosin-N-10 labels myofibrils, mPlum-γ-tubulin-N-17 labels centrosome, EGFP-β-catenin-N-7 labels the distribution of β-chain proteins, mApple-profilin-C-10 labels the distribution of anterior fibrinous, mKO-Pit1-N-6 labels the distribution of pituitary-specific transcription factors, and mEGFP-TPX2-N-10 labels the distribution of TPX2 proteins. Although this labeling method is simple and direct, certain problems still persist in applications. The typical problems are weak fluorescence signal, nonspecific aggregation and wrong location or interference of the function of the target proteins [57]. Therefore, we must consider whether the fluorescent protein we choose is appropriate, including whether the quantum yield is high, whether the monomer property is good, whether it is tolerable to photon-bleaching, whether it is sensitive to pH changes, or whether we should change the coupling positions (N-terminus, C-terminus, or other appropriate area) of the fluorescent proteins in the target protein molecules. If all of these problems have been solved, we should consider the expression quantity of fused proteins in the cells because these fusion proteins expressed by transient transfection are usually overexpressed under the control of constitutive promoters. In many cases, screening for stable cell lines with low expression or using inducible promoters is highly valuable work, which will not only improve the correctness and repeatability but is also convenient for quantitative analysis of experimental data [57].



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1.3.1.2 Characterization of protein spatial location and orientation (1) Labeling specific structures and organelles in cells We can usually find one or more proteins that are specifically located in specific structures or organelles in cells via a literature review, and we can track the structures or the organelles through the coupling of such proteins with fluorescent markers. Thus, we can determine the location of the labeled area, morphology, motion, and transformation of organelles, and the relationships of these organs with other structures also can be observed. For example, Saffarian et al. [58] coupled DsRed to the C-terminus of Clathrin LCa and expressed them in different cells. Subsequently, these researchers defined or determined two different kinetic models of the endocytosis process mediated by clathrin through dynamic microscopic imaging. Finally, this group also solved a subset of the arguments on this issue from the previous literature. In addition, Kogure et al. [33] first developed a new large Stokes-shift protein Keima (λex/em: 440/620 nm) and combined it with EGFP, thus demonstrating that two fluorescent proteins could be excited simultaneously with a single wavelength laser. The Keima mutation, dKeima570, which has an emission peak of 570 nm, was also generated. Subsequently, Takako et al. attempted to realize simultaneous excitation of six different fluorescent proteins located in different subcellular structures in a single cell at 458 nm. CFP was located in the cell membrane, mMiCy was located in the endoplasmic reticulum, EGFP was located in the Golgi apparatus, and YFP was located in the microtubules. dKeima570 was located in the nucleus and mKeima was located in mitochondria [33]. In 2008, Wang et al. [59], respectively, labeled the Racl (membrane structure), Golgi apparatus, endoplasmic reticulum, mitochondria, and nucleus with mCerulean (λex/em: 435/476 nm), EYFP (λex/em: 516/529 nm), RFP2 (λex/em: 588/625 nm), RFP1 (λex/em: 451/615 nm), and Hoechst33342 to successfully realize five-color fluorescence imaging in a single cell. (2) Simultaneous monitoring of the motions of multiple proteins in one event Certain events in cells, such as the process of autophagy generation, are the result of teamwork among many different proteins and molecules, and dynamic molecular information in the process of autophagy formation can be detected by labeling different proteins simultaneously. Hailey et al. [60], respectively, labeled LC3 and Atg5 with CFP and YFP to observe their recruitment via autophagic vesicles and understand their functions. The Donald C. Chang lab labeled Bax with CFP and labeled Bak or Smac with YFP to study the kinetic characteristics of multiple apoptosis-related proteins during cell apoptosis. These researchers found that Bax was distributed in the cytoplasm, Bak was distributed on the mitochondrial membrane, and Smac was distributed in the mitochondria in the absence of apoptosis [61]. In contrast, the following events occurred during the process of apoptosis: The Bak distributed on mitochondrial membranes gathered into spots and colocalized with Bax, which

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was transferred from the cytoplasm to the mitochondrial membrane, but Smac was released from the mitochondria to the cytoplasm. (3) Acquisition of protein dynamics and functional information In using fluorescent protein labeling methods to study the dynamic processes and functions of proteins in the cytoplasm or cell structures, corresponding fluorescence analysis techniques are also necessary, such as FRET, FRAP, and FRAPa [62]. Drake et al. [63] studied cytoplasm – nucleus exchange kinetics and the fluidity of EGFPLC3 in the cytoplasm and nucleus using FRAP. Kim et al. [64] studied the kinetics of Hsp70-YFP located in polyglutamine protein aggregates and demonstrated that Hsp70 was not bound to the protein aggregates, thus solving certain previous arguments on this issue.

1.3.2 Monitoring of gene expression in live cells Fluorescent proteins can be used as probes to monitor the gene expression levels in living cells. By constructing fluorescent proteins to the downstream of target promoters using genetic engineering, the expression of target genes can be monitored via the fluorescence signal, and the fluorescence intensity is positively correlated with the gene expression level. A fluorescent protein monomer is commonly used for direct labeling and tracking of target proteins because fluorescent protein multimers might interfere with the spatial location, fluidity, and interactions of target proteins. If we use the expression of fluorescent proteins to characterize the regulation and expression level of promoters in living cells, then we can choose fluorescent protein multimers, which are usually brighter than monomers and are more sensitive for gene expression monitoring. Therefore, many fluorescent proteins that have excellent physical and chemical properties are highly suitable for detection of gene expression, as evidenced by the following examples: (1) Detection of gene promoter expression level through fluorescent proteins: Chalfie et al. inserted a GFP gene downstream of the mec-7-lacZ promoter. By specifically inducing the expression of lacZ in nematodes, this group detected the generation and location of GFP [11]. (2) Monitoring of transcriptional timing of different promoters using fluorescent proteins of different colors: The Jen Sheen group separately fused sGFP (S65T) or GFP downstream of AtCAB2. Under variable light intensity stimulation, the AtCAB2 promoter in different tissues could be differentially activated, and this research was able to effectively define the effects of light stimulation on gene expression by measuring the fluorescent color difference in different tissues [65]. The Ann Tsukamoto group generated two GFP mutants (RSGFP4 and GFPS65T) with different spectra through mutation of the wtGFP. Using these two GFP mutations, the temporal and spatial differences of FACS gene expressions were



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monitored in human fibroblasts [66, 67]. (3) Detection of promoter temporal activity by fluorescent proteins: The timer fluorescent protein is a fluorescent protein that can alter color with passage of time, from blue to green, and finally to red. Thus, the time span of promoter activation could be measured via detection of the colors of the timer fluorescent protein spectra [54, 55]. In addition, the application of rapidly folded proteins would shorten the time between promoter activation and fluorescent signal emergence. BiFC, which is based on fluorescent proteins, has been recently used to detect promoter activity. BiFC is one of the protein complementary technologies in which the fluorescent protein is divided into two fragments at a specific site and expressed independently. Under specific conditions, the two fluorescent protein fragments can be spontaneously fused into a complete fluorescent protein and emit fluorescence (see the details of principles and applications in Section 1.6). Thus, if the two fluorescent protein fragments were separately constructed under the control of two different promoters, the fluorescent signal can be detected only after both promoters were activated [68].

1.3.3 Biological applications of photon-switchable proteins and photon-activatable proteins Photon-activatable proteins are fluorescent proteins that have a specific photosensitive property and can be transformed from dark to light or transformed between different wavelengths when illuminated by a specific laser. In the process of imaging, we can realize progressive activation of the photon-activatable protein molecules and rapid differentiation between the activated and the inactivated molecules. These fluorescent proteins offer advantages for three important applications: molecular and cell movement tracing, enhancement of SNR, and super-resolution fluorescence imaging.

1.3.3.1 Tracking of protein movement Using PA-FPs, tracking of protein movement in living cells with long duration and dynamic tracing can be achieved. To dynamically monitor protein movement, we first photoactivate the region of interested and subsequently track the activated PA-FPs with methods such as FRAP. In contrast to traditional FRAP, imaging methods based on PA-FPs are more direct and more convenient and can offer more information. In addition, these methods can be used for more rapid protein movement tracking. The irreversible PA-FPs can be used to monitor the movement of proteins in living cells, although they should not be used to activate and track repeatedly. These materials are convenient for long-term tracking of proteins due to their steady light signal,

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and they have become an effective complement to the dynamic monitoring method based on the photo-bleaching effect [69]. Compared with irreversible PA-FPs, PA-FPs could provide additional information in design of experiments and could offer more information or multiple time points as well as multiple spatial regions or orientations [70].

1.3.3.2 Tracking of cells, organelles, and subcellular structures Irreversible PA-FPs can be used to optically label cells in culture systems and tissues or in the whole body and can be applied to observe the motion and migration of the cells during the processes of development, carcinogenesis, and inflammation [71–73]. Danny A. Stark and Paul M. Kulesa first applied PA-FPs in intact chicken embryos and introduced PA-FPs into chicken neural tubes via electroporation. These researchers subsequently used confocal or two-photon microscopy to photoactivate and perform long-term studies on specific cells or neural crests. Moreover, these researchers carefully compared the advantages and disadvantages of different PA-FPs, such as PA-GFP and PS-CFP2 and KikGR and Kaedein labeling of living cells in tissues. Among these choices, PS-CFP2 was still visible 48 hours after photoactivation [74]. By constructing a fused plasmid that contains organelle position signals, PA-FPs could be used to label all of the organelles in live cells and track the movement and migratory direction of a single organelle as well as its interactions with other cells [75]. In addition, protein movements in organelles and protein exchanges in organelles or between different areas in cells also could be tracked using similar methods.

1.3.3.3 Monitoring of protein degradation Irreversible PA-FPs can be used to monitor the degradation of proteins. Because irreversible PA-FPs are different from ordinary fluorescent proteins with stable signals, the photon-activated signals of irreversible PA-FPs are independent of newly synthesized proteins. Therefore, if the photon-bleaching phenomenon can be avoided or corrected, changes in the photon-activated signal would be related only to the degradation rate of fusion proteins. This technique can be used to accurately and directly monitor the degradation of specific proteins in living cells [76]. Lukyanov et al. fused green-red photon-switchable protein Dendra2 to proteins whose degradation process was typically controllable (such as IκBα) and studied the protein degradation pathway by observing the red fluorescent signals in cells after photon activation. This method can be used to monitor the degradation process of IκBα more accurately and independent of newly synthesized proteins and to study the changes in protein degradation rates before and after drug stimulation.



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1.3.3.4 Enhancement of SNR Reversible PA-FPs can be switched between the dark state and light state via photon activation, whereas other fluorescent signals, especially background fluorescent signals, are relatively constant in biological imaging. Therefore, if we can separate the fluorescent protein signal from the background noise based on this significant difference, then high-contrast fluorescence imaging of specific structures in a high background environment can be achieved [77]. In 2008, Gerard Marriott et al. used optical lock-in detection imaging microscopy to distinguish a photon-controllable fluorophore from a background signal that could not be regulated by light, thus greatly improving the imaging contrast and detection of trace molecules. In their research, this group succeeded in labeling Dronpa to actin in NIH 3T3 and toad embryos as well as the nerves and muscles cells of zebra fish seedlings and obtained high-contrast images.

1.3.3.5 Super-resolution fluorescence imaging Near 2005, the super-resolution imaging technology based on PA-FPs, named after PALM and also known as PALMIRA [78] or FPALM [79–81], launched a revolution in the optical imaging field. By photon-controlling the luminous state of PA-FPs, we can obtain a fluorescence signal with a controllable sparse distribution, thereby defining the position precisely, and we can subsequently rebuild an optical image whose resolution is beyond the diffraction limit (200 nm). Currently, PALM is one of the most eagerly awaited super-resolution imaging technologies. Because the imaging accuracy of PALM is directly dependent on the quantum yield of the labeled fluorophore and the stability of the photoactivation state, PA-FPs play an important role in this technology. Recently, the highest localization accuracy of optical imaging systems based on intracellular fluorescent protein labeling reached 10 nm. Many single PA-FPs, whether reversible or irreversible, have been used in PALM. Thus far, PA-GFP [80, 81], Dronpa [82], PS-CFP2 [82], mEos2 [83], Dendra [83], and PA-mCherryl [41] have all had successful applications. The gradual emergence of mature technologies and additional optimized fluorescent proteins have made the imaging and tracking of many important cellular structures drop below the threshold of the optical limitation, and these imaging targets include microfilament and microtubule structures in the cytoskeleton, grid and spot structures in vesicular trafficking and a variety of functional protein complexes. This development has led to a deeper understanding of and research on the important physiological behaviors of the cells, such as cell migration, generation of immune synapse, and development of nerve cells. Different PA-FPs with spectra and photon-controlled processes that could be easily distinguished can be combined and applied in two- or three-color

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super-resolution imaging [84], and among them, the combination of PS-CFP2 and green and/or red photon-switchable proteins (mEOS family or Dendra2) is the most mature. Using two-color PALM, we can directly observe interactions between two different important proteins at a resolution of less than dozens of nanometers and define the extent of co-location of target proteins with other proteins or organelles [85]. For example, Subach et al., respectively, labeled Clathrin-LCa and transferrin receptor (TfR) with PA-GFP and PA-mCherry1 and detected the co-location of these structures using double-color PALM. These researchers found that TfR-PA-mCherry1 spots and PA-GFP-CLC spots presented three separate or closely adjacent structures under this super-resolution. This investigation revealed that the speckle characteristics and sizes that were obtained in this statistical analysis in different contact stages of TfR and CLC were consistent with the results gained from electron microscopy [41]. Soon afterwards, Samuel T. Hess used similar ideas to further clarify the co-location relationship between the TfR protein and the actin skeleton and membrane structure. These researchers selected three PAFPs (Dendra2, PamKate, and PA-mCherry1) and combined a sophisticated spectral resolution algorithm to finally realize three-color super-resolution imaging at the cellular level [84].

1.4 Functional fluorescent protein probes Because of the close relationship among molecular function, location of the molecule and the duration of its function, we need a long-term dynamic study of the functions of biological molecules in living cells. Because of their endogenous nontoxicity and accurate positioning ability in the cells, fluorescent protein probes have been widely used in dynamic monitoring of biological molecules in living cells. This chapter introduces redox probes, adenine nucleotide (ATP) fluorescent protein probes, pH probes, voltage-sensitive probes, calcium probes, mercury ion probes, copper ion probes, and zinc ion probes.

1.4.1 Redox probes Redox state plays an important role in cell proliferation, cell differentiation, cell aging, and cell death. The process of intracellular redox change is quite short; for instance, ROS can be found in the regulation of normal growth factor signaling and in longevity or aging cells. Redox events can change the osmotic pressure of mitochondria and release cytochrome C to induce cell death. The last process in cell apoptosis is breaking of the oxidation – reduction equilibrium. Therefore, it is highly important to develop redox probes that can monitor cellular redox status dynamically to



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understand the process of intracellular redox processes. Currently, certain researchers have modified fluorescent proteins and used them as probes to detect the redox state in the cells. Thus far, four types of probes are available: roGFPs, Grxl-roGFP2, HyPer, and rxYFP.

1.4.1.1 roGFPs roGFPs are mutated from A. victoria GFP. In roGFPs, the amino acid residues in the surface of the GFP tubular structures were replaced with a pair of cysteines. The pair of cysteines can form a disulfide bond in an oxidizing environment, and the disulfide bond unfolds in a reductive environment. Accordingly, by detecting the ratio of fluorescence intensity changes activated by excitation light with different wavelengths, the dynamic changes of the redox state in living cells can be rapidly and irreversibly reflected. This ratio probe can avoid the measurement errors caused by inhomogeneity of the probe concentration or photon bleaching of the probe. This ratio probe has two types: roGFP1 and roGFP2. roGFPs have two excitation peaks and one emission peak. The excitation peaks of roGFP1 are located at 400 nm and 475 nm, and the value at 400 nm is higher than that at 475 nm. Excitation of roGFP1 in the 405 nm and 488 nm bands is performed using an argon ion laser, and the received fluorescence has an emission peak at 510 nm. Therefore, we can track the changes of redox state by calculating the fluorescence intensity ratio of the emission peaks excited by a 405-nm laser and a 488-nm laser [86]. roGFP1 can reflect the changes in the mitochondrial redox state, which is induced by oxidation and reducing substances when it is located in the mitochondrial matrix in HeLa cells. Under normal physiological conditions of the HeLa cell, the changes in the redox potentials of mitochondria are within the detection range of roGFP1. HeLa cells expressing roGFP1 showed stronger reducibility when they were cultured in medium that contained glucose than in medium that contained galactose/glutamine. These results showed that the cells were forced to produce ATP via the tricarboxylic acid cycle when the culture medium simultaneously contained galactose and glutamine and the ratio of NADH/NAD+ decreased. These results suggested that the roGFP1 probe could be used to dynamically monitor the changes in the redox state in cells. roGFP2 was mutated from roGFP1. The S65T of roGFP1 was mutated, and the second excitation peak of roGFP1 was moved to 490 nm [87]. Moreover, the excitation peak at 490 nm is higher than that at 400 nm. roGFP2 has a wider dynamic range than roGFP1 for monitoring of redox potentials, and roGFP2 is more sensitive to pH value. roGFP2-PTS1 is a roGFP2 probe that locates in the peroxisomes of mammalian cells and can be used to monitor the redox state of peroxisomes. Additionally, it was found that the redox state of peroxisomes varied with the redox state of the environmental conditions.

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1.4.1.2 Grx1-roGFP2 Glutathione is the main redox buffer in cells; its redox potential is −240 mV, and its concentration varies from 1 mmol/L to 13 mmol/L. The sensitivity of traditional roGFP probe is not sufficient to monitor the changes of redox potential, and the roGFP probe cannot note the substance that influences the oxidation – reduction system. Based on this purpose, certain researchers developed one probe specifically used to monitor glutathione, known as Grx1-roGFP2 [88]. Grx1 is a small redox enzyme that can identify substrate glutathione and reduce or oxidize it. In the Grx1-roGFP2 probe, Grx1 can catalyze the rapid equilibrium between the redox pairs in glutathione and the redox pairs in roGFP2, and thus, the ratio spectrum of Grx1-roGFP2 can be used to reflect the concentration of glutathione. Grx1-roGFP2 does not change the sensitivity and spectral property of roGFP2. It is able to rapidly and sensitively detect glutathione concentrations in a living cell, so that the change in the redox state of the cells could be indirectly reflected [89]. The Grx1-roGFP2 probe can monitor nanomolar concentrations of oxidized glutathione GSSG and millimolar concentrations of reduced glutathione GSH in cells within a few seconds to a few minutes.

1.4.1.3 HyPer ROS play an important role in selected malignant diseases and in the process of severe inflammatory reactive ischemia and hypoxia. Additionally, H2O2 is a kind of ROS. Certain researchers inserted the regulatory region of the H2O2-sensitive protein OxyR into circular permuted YFP (cpYFP) and developed the HyPer [89] probe, which is specific to H2O2. The probe was named after the English peroxide hydrogen, which indicated that it was dedicated to H2O2 and was not sensitive to other oxides. In the presence of H2O2, reduced OxyR transforms to oxidized OxyR. The major amino acid residues of OxyR in this region are Cys199 and Cys208. When the probe is exposed to H2O2, Cys199 is released from the hydrophobic structure and forms a disulfide bond with the Cys208. Thus, the property of the cpYFP spectrum was changed. The probe has two excitation peaks located at 420 nm and 500 nm and one emission peak at 516 nm. When the concentration of H2O2 increases, the excitation peak value at 500 nm gradually increases, whereas the peak value at 420 nm decreases. The amount of H2O2 can be reflected by calculating the ratio of the emitted fluorescence intensity values generated by the excitation at 500 nm and 420 nm. To identify the sensitivity of HyPer to H2O2, researchers added H2O2 with different concentrations into suspended cells that expressed HyPer and found that the effect of H2O2 on the cells could be detected by HyPer even the concentration of H2O2 was less than 5 μmol/L. Under induction of the proapoptotic protein Apo2 L/TRAIL and NGF, the increase in the H2O2 concentration in the cytoplasm and mitochondria of single mammalian cells that expressed the HyPer probe could be detected.



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1.4.1.4 rxYFP The YFP-based redox probe referred to as rxYFP was screened by mutating the 149 and 202 sites of the beta barrel structure of YFP to Cys149 and Cys202 and was subsequently expressed with the downstream PGK1 promoter, thus creating a non-­ratio  –  type probe. The excitation and emission peaks of rxYFP are located, respectively, at 512 nm and 527 nm. rxYFP is a redox probe that is used relatively early to detect intracellular glutathione. The change of GSSG/GSH redox ratio can be detected; when thiol oxidant DTT-4 and reducing agent DPS were added in yeast cells that expressed rxYFP, the concentration of oxidized glutathione in the yeast cells was detected at 4 μmol/L. However, this probe has its limitations; it is not a ratiometric probe and its spectrum is similar to that of cellular autofluorescence, and therefore, it must be carefully screened before use. This protein was rarely used when isometric probes were invented.

1.4.2 ATP fluorescent protein probes ATP is the main energy substance in living cells and is also a signal molecule used to regulate energy site reactions, especially in regulation of ion channels and activation of the signaling cascade. Therefore, it is particularly important to detect the dynamic distribution of ATP in living cells under physiological conditions. Researchers have invented many probes for ATP detection that were used to elucidate how cells consume ATP to influence their physiological reactions. Early ATP probes were based on luciferase. The luminescence intensity of luciferase indirectly reflects the concentration of intracellular ATP because the radiant energy of luciferase is ATP. A concentration of ATP as low as 10−9 mol/L can be detected in vitro using this method [90]. However, if the ATP concentration is notably low in living cells, it is beyond the detection range of this probe for ATP concentration. M. Willemse developed an FRET probe to detect ATP in living cells. This probe contains a central domain of type II domain inosine monophosphate dehydrogenase (IMPDH2) that can be identified by ATP, and two ends of the central domain are connected with CFP and YFP. When the concentration of intracellular ATP increases, the fluorescence ratio of YFP/CFP decreases because FRET probes based on the IMPDH2 recognition domain are cut off. This type of probes can be used to dynamically monitor the concentration of ATP in the range of 1–20 mmol/L in cells [91]. Based on this information, certain researchers constructed a CFP-xa-YFP probe (xa, Xa protease sensitive cleavage sites) with a CFP/YFP FRET pair and studied the intracellular concentration of ATP via the fluorescence lifetime [92]. A higher concentration of intracellular ATP lead the CFP-Xa-YFP probes to be cut off more efficiently, resulting in an increase of the fluorescence lifetime of the donor CFP in the probes. A concentration of ATP as low as mmol/L can be detected using this

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method. The disadvantage of this probe is that it is unable to dynamically detect the variations in ATP. To create a more sensitive probe for detection of ATP, researchers used a circular permuted YFP Venus (cpmVenus) as a matrix probe. The cp fluorescent protein refers to a fluorescent protein whose original N-terminus and C-terminus are connected by a flexible polypeptide, and a new set of N-terminus and C-terminus is reopened near the other end of the chromophore. The area between the original N-terminus and C-terminus of cpVenus can be connected by a bacterial regulatory protein-GlnK1, which is combined with ATP. GlnK1 is a membrane protein of the PII family. This probe was optimized to detect the intracellular ATP/ADP ratio, and it was named GlnK1-cpmVenus QV5. This probe specifically detects intracellular ATP and ADP, whereas other nucleotides such as AMP or GTP or NAD have no effect on it. The excitation peaks of this probe are located at 490 nm and 405 nm, and the emission peak is at 530 nm. Therefore, intracellular ATP or ADP concentration can be characterized by the fluorescence intensity ratio of 488 nm and 405 nm laser excitation (FI488nmEx/FI405nmEx). The detectable concentrations of ATP and ADP are, respectively, 0.04 μmol/L and 0.2 μmol/L, and thus, ATP has five times the ADP affinity to GlnK1-cpm Venus QV5 [93]. With this probe, researchers tested the concentration of ATP and Ca2+ in islet β cells and illustrated the synthesis process of mitochondrial ATP in islet β cells and the functions of MCU (mitochondrial Ca2+ uniporter) [94].

1.4.3 pH probes pH probes refer to intracellular pH-sensitive probes. Currently, fluorescent protein-based pH probes primarily include two categories: fluorescent ratio probes and fluorescence intensity probes. By introducing a specific localization sequence, pH probes can be located in different subcellular structures, such as the Golgi, mitochondrial matrix, mitochondria membrane, and endoplasmic reticulum, and the pH values in different subcellular structures can be measured by the corresponding pH probes. For example, EGFP (pKa = 6.15) is used to test for pH value in acidic organelles, whereas EYFP (pKa = 7.1) is more suitable for detection of the variation of pH values in the mitochondrial matrix where the pH value is approximately 8.0. As early as in 1997, Patterson et al. noted that the fluorescence intensity of the wtGFP and its mutants decreased following the declining pH value if pH was less than 7 [95]. Hanson et al. [96] revealed that protonation in the vicinity of the chromophore results in changes in the hydrogen bonds, and thus the fluorescent protein changes in response to different pH values. Thereafter, researchers developed many new and excellent pH probes, thus providing a great tool for scientific research. However, pH probes based on the change in fluorescence intensity of fluorescent proteins have



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certain limitations in that they are easily influenced by autofluorescence and imaging conditions. In 1998, Miesenbock et al. screened the pH probe pHluorin based on ratio detection by mutating E132D, S147E, N149L, N164I, K166Q, I167V, R168H, S202H, L220F, and nine other sites of wtGFP. The probe exhibited reversible change when the pH was between 5.5 and 7.5, the response speed was faster than 20 ms, and the emission peaks at 395 nm and 475 nm, respectively, decreased and increased following the increasing pH value. This probe can be used to monitor vesicle secretion and synaptic transmission when it is connected with vesicle membrane proteins [97]. In 2011, Morimoto et al. [98] introduced the M153R mutation to pHluorin, thus greatly improving the connective stability of pHluorin and the fused proteins and solving hydrolysis of fused proteins while maintaining the other characteristics of the original probe. Hanson et al. proposed a new pH probe-deGFP, which is a mutant of GFP with two emission peaks located at 460 nm and 515 nm. When the pH decreased, the emission peak at 515 nm decreased and the emission peak at 460 nm increased. Therefore, changes in the intensity ratio of the two emission peaks can offer a good indication of changes in pH [96]. Four series of peGFP are available, namely, deGFP1, deGFP2, deGFP3 and deGFP4, and they differ mainly in the mutated position and the responses to pH values. Awaji et al. [99] constructed double-excitation and double-emission mode probes GFpH and YFpH by combining pH-sensitive GFP mutants (GFP or YFP) with a pH-insensitive GFP (GFPuv), thus characterizing pH changes by calculating the ratio of the emission peaks excited by 380 nm and 480 nm. In 2008, Urra et al. constructed a similar pH probe pHCECSensor01 by combining the pH-sensitive EYFP and pH-insensitive ECFP with the membrane protein displayed sequence, positioning the probe in the basement membrane. The pKa of pHCECSensor01 was 6.5 ± 0.04. It can be used to detect extracellular pH changes in epithelial cells or other structures [100]. In 2004, Abad et al. transformed the classical calcium probe “Camgaroos,” including the use of truncated 73 amino acid sequences in a jellyfish light-emitting protein to replace the original calcium response component in the Camgaroos probe, and added the mitochondrial targeting sequence. The newfound probe was named mtAlpHi [101]. The probe has a higher pKa value (approximately 8.5), the pH response scope ranges from 7 to 10.5, and its response to Ca2+ was eliminated. The probe can be used for detection of pH changes in organs with higher pH values (such as the mitochondrial matrix). In 2012, Ogata et al. [102] fused H148V-mutated Venus with GFPuv, added cytochrome C localization sequences, and finally constructed MTpHGV, a new pH probe for detection of mitochondrial pH. This probe was used to detect pH changes of mitochondria in pancreatic β cells and to study the role of mitochondria during insulin secretion. Common pH-sensitive fluorescent protein probes are shown in Table 1.1.

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Table 1.1: pH-sensitive fluorescent protein probes. Probe name

Fluorescent protein

pH detection range

Reference

pHluorin pHluorin-M153R deGFP GFpH YFpH pHCECSensor01 mtAlpHi MTpHGV

GFP mutant GFP mutant GFP mutant GFPuv/GFP GFPuv/YFP CFP/YFP YFP GFPuv/Venus

5.5–7.5 5.5–8.5 6.0–8.0 5.0–8.0 5.0–8.0 5.5–8.6 7.0–10.5 6.0–10.0

[97] [98] [96] [99] [99] [100] [101] [102]

1.4.4 Voltage-sensitive probes 1.4.4.1 First-generation voltage-sensitive probes As introduced in Baker et al.’s [103] review in 2008, the first-generation voltage-sensitive probes based on fluorescent proteins were constructed by combining fluorescent proteins with inophorous proteins controlled by voltage. Three main types of first-generation voltage-sensitive probes are: (1) FlaSh invented by the Isacoff laboratory, which was constructed by connecting the C-terminus of a wtGFP protein to the Drosophila Shaker K+ inophorous proteins [104]. When the probe is expressed in oocytes, a voltage change of −80 mV depolarization results in an approximate 5 % decrease of the GFP fluorescence intensity. Guerrero et al. [105] developed different characteristics of FlaSh probes with a higher SNR using different fluorescent protein chromophores to replace wtGFP in the FlaSh probe, thus providing additional options for action potentials or synaptic potential detection. (2) VSFP1 invented by Knopfel laboratory is a voltage-dependent conformation-change FRET probe constructed by linking two ends of the quaternary transmembrane sequences in Kv2.1 (a voltage-gated K+ channel protein) with CFP and YPF [106]. (3) SPARC was invented by the Pieribone laboratory by inserting a fluorescent protein into the gap of domain 1 and domain 2 of rat skeletal muscle Na+ inophorous protein to monitor membrane voltage changes [107]. Because of poor membrane expression characteristics in mammalian cells, the first-generation voltage-sensitive probes had limited applications. In addition, a large number of nonresponsive background signals also largely overshadowed the voltage-dependent signal.

1.4.4.2 Second-generation voltage-sensitive probes The discovery of Ciona intestinalis voltage sensor phosphorylated protein (CiVSP) greatly promoted the development of voltage-sensitive probes. Ci-VSP is a



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voltage-controlled enzyme protein that contains a transmembrane voltage-sensing domain and a cytoplasmic kinase phosphatase domain, and it has a domain similar to the S1–S4 quarternary transmembrane domain in K+ inophorous protein. The expression of this probe does not affect proper functioning of cells compared with that of inophorous voltage-sensitive proteins, and therefore, there is no need to control its expression level in the cells. VSFP2 is a Ci-VSP receptor protein-based voltage-sensitive probe; it is an FRET signal detection probe based on CFP and YFP and was also first invented in the Knopfel laboratory [108]. The VSFP2 probe showed a strong fluorescent signal and could be well located in the plasma membrane when it was expressed in PC12 cells or hippocampal neurons cells. VSFP2.1 was obtained by R217Q point mutation of Ci-VSP in VSFP2; it has stronger membrane localization ability and faster signal response to membrane potential [108]. VSFP2.1 is well suited for the detection of neuronal activity, such as large synaptic voltages and single-action potential signals.

1.4.4.3 Third-generation voltage-sensitive probes The third-generation voltage-sensitive probes were transformed primarily in the following directions: optimization of connection sequences, use of different fluorescent protein mutants, and a new method of fluorescent protein fusion with voltage-sensitive proteins. Although previous VSFP mutants showed good membrane localization and obvious dynamic range responses, the response of the fluorescent signal change to the voltage change was slow and not sufficient to satisfy demand for determination of neuronal fast voltage signal changes. VSFP2 uses an FRET signal based on YFP and CFP to characterize voltage changes, and although the Knopfel laboratory had demonstrated that the receptor did not necessarily require the presence of YFP, if the voltage changes were directly characterized by a CFP signal, it would improve the response speed of the probe, and this new probe was named VSFP3.1 [109]. When the probes were transfected into cells, the signals were detected by receptor-bleaching FRET, and thus, the signals were completely free from receptor influence. Furthermore, the experiments also showed that the response speed of the probe had increased to 1.3 ± 1 ms. Based on VSFP3.1, CFP was replaced by with Cerulean, Citrine, mOrange2, TagRFP, or other mutant proteins of mKate2. The results confirmed that VSFP3.1_mOrange2 probe exhibited better voltage signal characterization ability in hippocampus neuron cells [110]. Red-shift fluorescent proteins greatly broaden the applied scope of membrane voltage probes and also provide many spaces for combination with other non-red-shift probes. CFP and YFP are the respective major donor and acceptor of FRET for VSFP2.1 and VSFP2.3, etc. Mutoh et al. used mCitrine and mKate2 as the FRET donor and receptor to construct a new voltage-sensitive FRET probe VSFP2.4, which has dynamics

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similar to those of VSFP2.3 [111]. The introduction of magenta protein mKate not only has expanded the spectral applied scope of the probe, but also, more importantly, avoids autofluorescence interference and provides viability for in vivo applications to a certain extent.

1.4.4.4 Other types of voltage-sensitive probes The first-generation voltage-sensitive probes have limited applications in living cells because of the characteristics of the sensor proteins, primarily poor membrane localization characteristics and high background fluorescence interference. Jing et al. [112] used fluorescent protein bimolecular complementary technology to randomly connect the two fragments of Venus fluorescent protein to Shaker protein through transposon technology. Tetramerized Shaker proteins are used to group fluorescent proteins and then emit fluorescence, thus improving probe sensitivity because it avoids cross talk from unsuccessfully folded Shaker proteins. Common voltage-sensitive probes are shown in Table 1.2. Table 1.2: Voltage-sensitive probes. Probe name

Sensor protein

Fluorescent protein

Reference

FlaSh VSFP1 SPRAC VSFP2.1 VSFP2.3 VSFP2.4 VSFP3.1 VSFP3.1_mOrange2 FlaSh-YFP

Shaker Kv2.1 Mouse skeletal muscle Na+ channel protein Ci-VSP Ci-VSP Ci-VSP Ci-VSP Ci-VSP Shaker

wtGFP CFP/YFP GFP CFP/YFP CFP/YFP mCtrine/mKate2 CFP mOrange2 Venus

[103] [106] [107] [108] [109] [111] [109] [110] [112]

1.4.5 Calcium probes 1.4.5.1 Calmodulin-based FRET probes The first reported calcium probe based on fluorescent proteins was cameleon-1, which consists of an FRET pair composed of BFP and GFP (S65T) located at the two ends, and a middle calcium sensor unit (i. e., calmodulin (CaM) C-terminus and its binding polypeptide M13) [113]. CaM and M13 are located near each other in the presence of Ca2+, which causes BFP and GFP to approach each other, and thus, FRET occurs. The intracellular calcium concentration can be characterized using the emission



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peak ratio of the donor and the acceptor. The FRET signals are positively correlated with Ca2+ concentrations, and the measurement range of this probe for Ca2+ is 10–7 to 10–4 mol/L. Because of the poor spectral properties of cameleon-1 BFP, the SNR is rather low in mammalian cell imaging. To overcome this shortcoming, Miyawaki used the classical FRET pair CFP/YFP to replace the BFP/GFP, and the new Ca2+ probe was named yellow cameleon2.1 (referred to as YC2.1) [114]. By virtue of the reversible binding of CaM-M13 and Ca2+, the YC2.1 probe can be used for real-time detection of Ca2+ concentration changes in living cells or in solutions. However, many shortcomings still exist in the YC2.1 probe. For example, its detection dynamic range decreased significantly when it was used in the cytoplasmic membrane in hippocampal neurons. This change was caused by the combination between large numbers of intracellular endogenous CaM with the M13 in the probe. The endogenous CaM not only could influence the sensitivity of the probe but the inherently high expression of CaM in the probe also could affect the function of endogenous CaM. Therefore, the Griesbeck and Tisen groups committed to reducing the binding capacity between the probe and endogenous CaM to optimize the YC series probes. Truong et al. [114] reduced the sensitivity of YC2.1 to pH changes through mutation of the probe, making it more conducive to expression and detection of calcium signals in the cytoplasm, and the new probe was named YC3.1. Subsequently, Miyawaki et al. constructed YC6.1 using a CaM-dependent kinase kinase (CKKp)-specific combination with CaM [115]. The FRET efficiency was tripled in living cell imaging, making it easier to observe the dynamic changes of Ca2+ in living cells. Using circular permutation (CP), fluorescent proteins such as cpYFP, new calcium probes (such as YC3.12 and YC3.20, YC3.30, and YC3.60), and YC3.90 have been generated by replacing the EYFP receptor in the original YC probe with a different  CP  Venus  (cpVenus) [116]. It has been found that the YC3.6 offers the best ­performance with an approximately 600 % dynamic detection range, greatly improving the dynamic range and acid stability and accelerating the calcium probe ripening.

1.4.5.2 Calcium probes based on TnC Heim and Griesbeck [117] discovered a novel calcium-binding protein troponinC (referred to as TnC) and constructed another FRET-based calcium probe by replacing the traditional CaM-M13 with TnC; the new probe was named TNL15. The probe did not combine with endogenous CaM, was undistrubed from CaM, and its expression does not affect the function of CaM in cells. The dynamic detective range of the probe was approximately 140 %, and it was successfully used for Ca2+ signal detection in neurons and myocardial cells.

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Thereafter, Griesbeck changed the receptor of the TN-L15 probe to fluorescent protein cpCitrine174, thus greatly improving the dynamic detective range of the calcium (from 140 % to 400 %); the new probe was named TN-XL. In 2008, Mank transformed TN-XL by removing the first two domains of TnC and repeating the latter two mutated domains, finally obtaining the new Ca2+ probe known as TNXXL [118]. TNXXL has higher detection sensitivity to calcium signals and is able to monitor the dynamic changes of Ca2+ signals in neurons in the mouse cortex for days or even weeks. In 2011, Liu et al. obtained TN-3XL by further optimization of TN-XXL; the CFP/ cpCitrine174 FRET pair in TN-XXL was replaced with a new FRET pair that consisted of a donor (a new CFP mutant created by random mutation) and cpVenus173 receptor, thus increasing the dynamic range up to 1156 %. The probe was applied in PC12 cells and showed that the dynamic range was significantly higher than that of the TN-XXL probe. The results suggested that the dynamic range of TN-3XL was significantly higher than that of TN-XXL in PC cells [119].

1.4.5.3 GCaMP calcium probes CP of fluorescent protein probes provides a new strategy for construction of a single fluorescent protein probe, and its application in calcium probes epitomized its unique advantages. GCaMP probes are calcium probes that are constructed using pEGFP, and the Ca2+ detective unit was also based on its interaction with CaM-M13. Unlike the FRET probes, M13 and CaM are, respectively, located at the N-terminus and C-terminus of cpEGFP. The pEGFP emission peak intensity increases when the Ca2+ concentration changes. The probe showed high affinity to Ca2+, and the affinity constant reached 235 nmol/L [120]. Because the fluorescence intensity of GCaMP is weak, GCaMP is easily influenced by pH. Therefore, Ohkura et al. implemented V163A and S175G point mutations on cpEGFP and harvested GCaMP1.6 probe, thus decreasing the pH sensitivity (the pKa was 8.2 when Ca2+ reached the saturation point) and also improving the selectivity of the response to Ca2+. Additionally, this group developed GCaMP1.6 (E140K) by further E140K point mutation in CaM, which not only improved the fluorescence intensity but also more importantly reduced the effects of intrinsic free Ca2+ on the probes [121]. Tallini et al. [122] continued relevant mutations based on GCaMP1.6 and introduced a protein sequence that would improve the heat stability to finally create the GCaMP2 probe. The fluorescence intensity of GCaMP2 was increased to six times that of GCaMP and GCaMP1.6, and the dynamic range was increased by four to five times. The most important outcome was a significant improvement in the stability of calcium probe at 37 °C such that it can be applied to calcium imaging in mouse heart cells and realize Ca2+ signal observation for 4 weeks. Tian et al. GCaMP continued to ameliorate GCaMP in 2009 by mutating M153K and T203V of cpEGFP as well as N60D of CaM to obtain GCaMP3. Eventually, the fluorescence was increased by approximately two times, and



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the dynamic range was increased by three times. The Ca2+ affinity increased by 1.3 times, and the new probe worked well in neuron imaging in worms, flies, and mice [123]. Douglas Kim and his colleagues obtained a new ultrasensitive GCaMP6 calcium probe via selective “mutation” and improved both temporal and spatial resolution in vivo (fly, zebra fish). The calcium signaling evoked in single-action potentials in vivo using GCaMP6 was 10 times faster than that of GCaMP3, and the dynamics were 2 times faster; thus, GCaMP6 triumphed over OGB-1 (a chemical calcium indicator). Moreover, GCaMP6 can reliably detect single-action potentials and a “single ridge direction adjustment” in the mouse visual cortex. The GCaMP6 sensor can be used for imaging a large number of neurons and tiny synaptic spaces for multiple times separated by a few months [124].

1.4.5.4 Camgaroo calcium probes Baird et al. constructed the Camgaroo 1 probe by inserting an exogenous CaM protein between the 144 and 146 sites of EYFP. The fluorescence intensity changed approximately 8-fold before and after Ca2+ binding [125]. However, for this non-ratio calcium probe, when the probe plasmids were transfected into cells and expressed, the calcium signaling in the quiescent condition was almost nil, making it difficult to use in cell experiment, and the protein expression level of this probe at 37 °C was not satisfactory. To correct this shortcoming, the Camgaroo 2 probe was created using Citrine, an EYFP mutant that showed better maturity at 37 °C. The fluorescence intensity changed by factor of approximately 6 before and after binding with Ca2+, and the affinity constant was approximately 5.3 µmol/L [126].

1.4.5.5 Pericam calcium probes Similar to the GCaMP probes, the pericam probes were also based on CP fluorescent proteins, except that GCaMP used cpEGFP, whereas pericam used cpEYFP, and M13 and CaM were, respectively, located at the N-terminus and C-terminus of cpEYFP. To achieve a greater dynamic range, a single amino acid was optimized to obtain several different pericam probes [127]. As a result, the dynamic range was expanded significantly after His203Tyr mutation in cpEYFP, and the fluorescence intensity increased by a factor of approximately 8. The new probe was named flash-pericam, and the folding efficiency of the probe at 37 °C was improved by mutating V163A and S175G simultaneously [127]. Flash-pericam was further mutated in H203F/H148D/F46L, and the Gly in front of CaM was deleted, whereas the original Link sequence (GGSGG) between the N-terminus and C-terminus of EYFP was replaced by VDGGSGGTG, thus producing a calcium probe that could be used to characterize the ratio of a doubleexcitation spectrum. The probe was named ratiometric-pericam [127]; its excitation

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spectrum changed with changes in Ca2+ concentration, its two excitation peaks were located at 495 nm and 515 nm, and the affinity constant was 17 μmol/L. Surprisingly, the emission intensity of pericam decreased by approximately 15 % under a 500-nm excitation wavelength after the D148T mutant was introduced into ratiometric-pericam and showed the exact opposite response of pericam to calcium, that is, the fluorescence intensity was lowest when the calcium was saturated but was strongest without calcium. The dynamic range changed by a factor of 7 before and after binding to calcium. Therefore, the probe was named inverse pericam [127].

1.4.6 Mercury ion probes Gu et al. first reported fluorescent protein probes used for Hg2+ detection. Near-IFPs became luminescent after its C24 cysteine residues were combined with biliverdin (BV); yet Hg2+ can undergo competitive binding to these residues, thus affecting the IFP fluorescence. In other words, the higher the Hg2+ concentration, the lower the fluorescence intensity of the IFP probe will be [128]. The detection limit of Hg2+ probe is approximately 50 nmol/L, and a concentration as a high as 1200 nmol/L does not reach the saturation state of the probe (detection range of 50–1400 nmol/L). This probe has high specificity to Hg2+ compared with other metal ions, and the pH adaptive range is wider. Mercury ion probes have similar responses to Hg2+ when pH is in the range of 5.5–8.8, thus providing effective tools for detection of Hg2+ in organisms and tissues. In addition, the agarose-protein – conjugated gel strips based on this principle have made it highly convenient to detect Hg2+.

1.4.7 Copper ion probes Sumner et al. [129] found that RFPs DsRed originated in tropical coral have high ­specific reversible affinity to Cu+ and Cu2+, and the detection limit was at the nanomolar level. The affinity constants for Cu+ and Cu2+ were 450 nmol/L and 540 nmol/L, ­respectively. The affinity of DsRed to Cu2+ was 7 orders of magnitude higher than that of wtGFP, and even with respect to certain mutants of GFP, the affinity of DsRed to Cu2+ was 40 times higher. In further studies, Rahimi et al. [130] found that histidine and cysteine residues played a key role in the binding process between DsRed and Cu2+. Using spectroscopic means analysis, Isarankura-Na-Ayudhya et al. found that His6-GFP fused with hisactophilin tags has high affinity to copper ions. The fluorescence intensity decreased approximately 60 % in 500 μmol/L Cu2+ solution, whereas the fluorescence intensity decreased by approximately 10 % and 20 % in Zn ion and Cd ion solutions. When EDTA was used to chelate Cu2+, the fluorescent intensity was restored to 80 % of the original value, suggesting that the probe has a good reversible



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binding to Cu2+ [131]. Further research revealed that the fluorescent intensity changes were primarily due to fluorescent molecular ground state quenching after binding to Cu2+ rather than changes to the fluorescent protein structure or conformation. When prepared, the His6-GFP gel-like test kit has a notably fast response to Cu2+ in solutions, thus achieving real-time detection of Cu2+ content, and the detection range spans 0.5 μmol/L to 50 mmol/L.

1.4.8 Zinc ion probes The first category of zinc ion probes was constructed based on single fluorescent proteins and transformed the amino acids near the fluorescent protein chromophore such that it would be sensitive to Zn2+. Barondeau et al. [132] reported the Zn2+ probe BFPms1 in which the 66 tyrosine of BFP chromophore was replaced by histidine (Y66H) to facilitate binding of metal ions, and the fluorescence intensity was approximately two times higher when Zn2+ was saturated. Mizuno et al. constructed cpGFP190-IZ-H using cpGFP, and its Zn2+ affinity constant was 570 nmol/L. Unfortunately, the probe responses to copper ion and nickel ion concentration changes were similar [133]. Because single fluorescent protein probes responded specifically to Zn2+, and the detection accuracy of fluorescence intensity detection technology was easily affected by photon bleaching, this probe was rarely used in quantitative detection of intracellular zinc ions [134]. The second category of Zn2+ probes consists of FRET probes based on FRET pairs. The fluorescent protein donor and acceptor are connected by the Zn2+ binding domain protein or polypeptide. Evers et al. constructed a Zn2+ probe (ZinCh-9) based on FRET using ECFP and EYFP as donor and receptor, respectively, and connecting the middle section with a Zn2+-binding polypeptide. When Zn2+ binds to ZinCh-9, the signal increases by approximately four times, and the Zn2+ detection range spans 10 nmol/L to 1 mmol/L [135]. Using a similar principle, Evers et al. constructed CLY9-2His by inserting a histidine (His) tag on both the N-terminus and C-terminus of the ECFPlinker-EYFP. The Zn2+ affinity constant of CLY9-2His was 47 nmol/L, and the Zn2+ sensitivity was improved significantly compared with ZincCh-9, but its dynamic detective range decreased, with only a 1.6-fold change in the fluorescence signal before and after Zn2+ binding [136]. Although ZinCh-9 and CLY9-2His showed good dynamic range and Zn2+ detection specificity, they were useless in high Zn2+ concentration areas because their detection ranges were below 1 mmol/L. Researchers have reported new Zn2+ probes based on FRET to ensure that the detection range of Zn2+ probes better matches the intracellular Zn2+ concentrations. Pearce et al. used human metallothionein (hMTIIa) as Zn2+-binding receptors; the two ends of hMTIIa were connected with ECFP and EYFP as the respective donor and the acceptor to build a Zn2+ probe FRET-MT [137–139]. Unfortunately, the affinity and specificity to Zn2+ were not satisfactory. Thompson and

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coworkers [140] used carbonic anhydrase (CA) as a Zn2+-binding receptor to construct a Zn2+ probe that has preferable affinity and specificity to Zn2+. In addition, Zn2+ detection probes can be constructed using zinc finger domains. Qiao et al. [141] fused ECFP and EYFP to ZF1/ZF2 or ZF3/ZF4 (two zinc finger domains from yeast) to generate two probes that can be used for the detection of Zn2+ concentration. Dittmer et al. used a similar Zn2+-binding receptor Zif268 to construct the FRET probes Cys2His2 and His4, which can be used to detect cytoplasmic and mitochondrial Zn2+ concentrations in single cells [142]. Although the probe signal changes by a factor of 2–4 in vitro but only changes 0.25 times within living cells, the probe improved the Zn2+ detection range. The Zn2+ affinity constant of Cys2His2 is 1.7 μmol/L, and in His4, it reaches 160 μmol/L, making it possible to detect Zn2+ concentrations in living cells. Interestingly, by far the best Zn2+-binding receptors are ATOX1 and WD4 associated with copper ions. Merkx et al. developed the CALWY series Zn2+ detection probes. After a series of modifications, the dynamic range was improved from 15 % to 200 % [137–139]. Subsequently, the eCALWY series probes were obtained via point mutation on the C416S site of the Zn2+-binding receptor of CALWY, which completely eliminated the binding of copper ions, and the dynamic range exceeded 200 %. The eCALWY series probes can be used for intracellular Zn2+ concentration detection.

1.5 Fluorescence resonance energy transfer (FRET) probes 1.5.1 Introduction of FRET 1.5.1.1 FRET measurement methods FRET technology has been widely used in biophysics and biochemistry. With the combination of FRET with fluorescence microscopy, biomolecular location and molecular dynamics can be accurately determined, including protein – protein, protein – DNA interactions, protease activity, and FRET efficiency changes caused by protein conformational changes [143]. FRET efficiency is primarily measured using the following methods: (1) FRET imaging based on fluorescence intensity: To determine whether two biomolecules (DNA – protein or protein – protein) approach and interact with each other, we can conjugate the fluorescent protein donor and acceptor to the two biomolecules; when the distance between the two biomolecules is 1–10 nm, FRET occurs. In other words, a portion of the energy of the donor molecule transfers to the receptor molecule such that the emission intensity of the receptor molecule is enhanced. In general,



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the donor molecule and acceptor molecule are connected to the respective ends of a single molecular probe to detect conformational changes of the probe. When the probe conformation changes due to stimulation from external factors, the spatial location and dipolar orientation of the donor molecule and acceptor molecule will change accordingly to generate or eliminate the FRET phenomenon. If the interactions of or conformational changes in protein molecules depend on ligand binding, then FRET might be highly suitable for detection of molecular behaviors of the ligand [144, 145]. (2) FRET imaging based on photon bleaching: FRET can also detect the fluorescence intensity change ratio after bleaching of the donor in the presence or absence of receptors [146, 147]. (3) FRET imaging based on fluorescence lifetime: The fluorescence lifetime is defined as the time delay ratio from fluorophore excitation to fluorescence emission. Fluorescence lifetime is an inherent characteristic of fluorophores, and it is not affected by the excitation light intensity or probe concentration, but the cellular microenvironment (e. g., pH value and ionic concentration) can influence the fluorescence lifetime. FRET imaging based on fluorescence lifetime (FRET  –  FLIM) is mostly used to detect donor fluorescence lifetime changes in the presence or absence of receptor molecules [148].

1.5.1.2 FRET pairs based on fluorescent proteins FRET probes usually consist of two fluorescent proteins with different spectra, and the two fluorescent proteins act as donor molecule and acceptor molecule. FRET probes based on fluorescent proteins have certain advantages over FRET pairs that consist of organic dyes: (1) they are biocompatible and innocuous to the target proteins or cells, and (2) they can be stably expressed in cells via genetic methods, and the fluorescence intensity will not decrease with the cell division. The disadvantages are that the excitation and emission spectra of FRET probes based on fluorescent proteins are wider than those of FRET pairs based on organic dyes. Thus, we need to set a series of controls to eliminate the influence of spectral cross talk on FRET efficiency calculation in FRET imaging. However, it is also difficult to apply this technique simultaneously with other FRET pairs to perform two-FRET detection in living cells. BFP and GFP formed the fluorescent protein pair first used in FRET. However, the initially selected GFP was easily photon bleached, and BFP had a low quantum yield. In addition, the excitation peak of BFP was located in the ultraviolet region, which was easily interfered by cellular autofluorescence. CFP and YPF have become the most commonly used FRET pair, although new fluorescent protein mutants are continuously produced, and FRET probes consisting of a CFP and YPF pair have high detection sensitivity due to high FRET efficiency. Subsequently, a variety of FRET pairs consisting of CFY and YFP mutants with better physical and chemical properties have further improved the dynamic detection range and detection sensitivity of FRET probes (Tables 1.3 and 1.4) [34].

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Table 1.3: Major fluorescent protein FRET pairs. Donor

Receptor

Description

Reference

mTagBFP

sfGFP

[149]

ECFP

Cerulean T-Sapphire

EYFP/Citrine/ Venus mDsRED/ tdDsRED EYFP DsRED

A blue/green FRET pair that is most suitable for ratio imaging. This pair is suitable for all FRET detection technologies.

mVenus

mKOκ

ECFP/EGFP

This pair has better spectral discrimination than the CFP/YFP pair. A CFP mutant more suitable for FLIM. This pair has the best spectral discrimination currently but requires UV excitation A red-shift FRET pair that can be imaged with GFP at the same time in the same cell with notably low cross talk.

[126, 150] [151] [152] [31] [153]

Table 1.4: FRET fluorescent protein pairs for multiratio imaging. FRET pair1

FRET pair2

Reference

CFP/YFP

mOrange/mCherry

[154]

mTFP1/YFP

mAmetrine/tdTomato

[34]

mVenus/mKOκ

mTagBFP/sfGFP

[149]

1.5.2 FRET imaging in cell biology research 1.5.2.1 Study of nucleic acids FRET has been used to analyze the structure of chromosomes and DNA. The FRETbased homologous DNA diagnostic analysis technique is able to monitor primer extension under the guidance of a template and has also been used for high-throughput genome analysis. Moreover, FRET-based chromosome fluorescence in situ hybridization is another important application. The underlying principle is that nucleic acids and probes for detection are labeled with different fluorescent molecules, and these two types of fluorescent molecules act as the energy donor molecule and acceptor molecule of FRET. Changes in fluorescence intensity reflect the spatial distance between the probe and the target gene. FRET occurs when the probe and the target gene combine with each other and their conformations change; otherwise, FRET disappears if the probe and the target sequence remain discrete. In addition, by labeling nucleic acids with energy donor and receptor pairs in different locations, FRET has expanded many aspects of nucleic acid studies, such as the study of DNA structure and regulation and degradation of nucleic acids [155].



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1.5.2.2 Study of protein structure and function FRET offers great advantages in the study of changes in protein structures and protein spatial distances [156, 157]. Compared with X-ray diffraction, FRET is able to analyze the dynamic processes of structural changes. In studies of protein interaction, FRET can monitor dynamic information within the target area in real-time compared with the classic yeast two-hybrid system and mammalian two-hybrid system. Pozo et al. used CFP/YFP as an FRET energy donor and acceptor pair in the study of interactions between integrin and their related proteins and found that integrin can mediate Rac protein translocation to a region near the cytomembrane and subsequently separate the inhibitory factor Rho-GDI from the Rac protein to activate the Rac protein.

1.5.2.3 Second messenger content monitoring FRET probes can detect changes in intracellular small molecule concentrations (such as calcium), and FRET probes also can be used to study the relationships between concentration and specific biological phenomena. Adams et al. reported the first FRET probe for detection of cAMP. BFP and GFP were linked by a specific polypeptide that can specifically identify cAMP-combined kinase. When the polypeptide was phosphorylated by the kinase (PKA), thereby changing the conformation of polypeptide, FRET occurred. Hence, kinase phosphorylation kinetics can be determined by the efficiency of the change process monitored by FRET probes [158, 159].

1.5.2.4 Protease activity detection A variety of proteases present in the cell, and FRET probes based on specific substrates of the proteases can effectively detect specific protease activity in cells. For example, Heim and Tsien selected a polypeptide with only 25 amino acid residues from trypsin substrates and used the peptide as linker to connect BFP (fluorescence donor) and GFP (fluorescent acceptor). Before trypsin activation, the small peptide maintained energy transfer from the BFP to the GFP, but after trypsin activation, the peptide was cleaved, BFP and GFP were separated from each other, and FRET gradually disappeared simultaneously because of the increased spatial distance [160, 161].

1.5.2.5 Study of cell apoptosis Xu et al. found that in all Caspase-3 substrates, DEVD was the most suitable specific FRET substrate for detection of Caspase-3. The FRET probe used DEVD-linked BFP and EGFP that could detect the activation of Caspase-3 during apoptosis. When

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Caspase-3 was activated, DEVD was gradually digested by Caspase-3, which separated BFP and GFP from each other, and FRET disappeared [162]. FRET probes for the major caspase enzymes in the apoptosis process have been developed, including Caspase-2, Caspase-8, and Caspase-9 [163, 164].

1.5.3 Intramolecular FRET probes Because the FRET signal is easily influenced by the spatial distance between the fluorophores as well as the dipolar orientation, the development of FRET probes with high sensitivity is limited. Crystal analysis results based on structural biology provided insights for developing and optimizing new FRET probes. However, the available FRET molecular probes are still limited at present and mainly rely on conformational changes or enzyme hydrolysis of internal specific substrates (linkers) of the probes to detect FRET signal changes [165, 166]. These probes include the Caspase detection proteases family [163, 164], secreted proteases (matrix metalloproteases, MMPs) [167, 168], and transmembrane proteases (β-secretase) [169–171] as the representative for enzyme FRET probes, and conformational change FRET probes for detecting the protein kinase family (such as Src, PKA, and Rac) [149, 172] and Ca2+ signaling [173].

1.5.3.1 Caspase probes Currently, 14 types of Caspase family proteases involved in apoptosis have been found. The Caspase recognition substrate in the probe is composed of at least four specific amino acid residues, and activated Caspase is able to cleave Asp-X in the substrate sequence. Different Caspases have different substrate recognition sequences. However, Caspase does not always cut those proteins that compose the tetrapeptide sequence, and it is possible that the multimer structural unit affects recognition of the substrate sequence. In the process of apoptosis, activation of different Caspase proteases occurs in strict compliance with the appropriate spatial and temporal characteristics. The specific substrates of Caspase family FRET probes have been reported as follows: the Caspase-1 recognition sequence is Tyr-Val-Ala-Asp (YVAD); the Caspase-2 recognition sequence is Asp-Glu-His-Asp (DEHD); the Caspase-3 recognition sequence is AspGlu-Val-Asp (DEVD), which can hydrolyze poly-ADP ribose polymerase (PARP); the Caspase-6 recognition sequence is Val-Glu-Ile-Asp (VEID), which can cleave lamins; the Caspase-8 recognition sequence is Ile-Glu-Thr-Asp (IETD); and the Caspase-9 recognition sequence is Leu-Glu-His-Asp (LEHD). Caspase-specific FRET probes can be built using the caspase substrate sequence (e. g., DEVD) as a linker to connect two fluorescent proteins with different spectral characteristics (e. g., CFP/YFP). Generally, when caspases in cells are inactivated, the fluorescence donor and fluorescence



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acceptor can approach each other, thereby producing the FRET phenomenon due to the presence of the linker. When caspases in cells are activated, they specifically cleave the internal restriction sites of the linkers in the FRET probes, the fluorescent protein pairs (such as CFP/YFP) are separated from each other, and the FRET phenomenon gradually disappears. Combined with microscopic imaging, full-time kinetic data can be obtained for Caspase activation under certain conditions. The Luo research group used CFP and RFP (DsRed) as the respective donor and acceptor of a FRET probe and connected the caspase recognition sequence (CRS) of Caspase-2 and Caspase-3 to  CFP and DsRed, respectively. As a result, this group constructed FRET probes (CFP–CRS–DsRed) for detection of the activation of the original caspase protease (Caspase-2) and effector caspase protease (Caspase-3). Thus, FRET can be applied to real-time dynamic monitoring of the chronological order and characteristics of Caspase-2 and Caspase-3 during apoptosis, discovery of Caspase-3-dependent independent pathways, coexisting phenomena in TRAIL-induced and Caspase-3-­ HeLa apoptosis, and visual and dynamic revelation of the relationship between the TK gene therapy, its lethal side effects, and the activation of Caspase-3 [163, 164].

1.5.3.2 Src probes In 2001, Tsien et al. used ECFP and EYFP to form an FRET pair using the SH2 sequence and EIYGEF sequence as the FRET probe linker. The ratio of the FRET probe changed by more than 25 % under stimulation of platelet-derived growth factor or epidermal growth factor (EGF). The results from a subsequent experiment demonstrated that in addition to Src kinase, the probe also responded to epidermal biological factor receptor (EGFR). In 2005, Shu Chien et al. transformed the poorly specific Src probes using the WMEDYDYVHLQG sequence in p130cas as linker, and thus greatly enhanced the specificity of Src probes. The modified Src probes were applied to dynamically monitor the rapid activation of peripheral Src in human umbilical vein endothelial cells and to quantitatively study the diffusion rate of Src in the cell membrane. In 2008, Wang et al. enlarged the dynamic range of Src probe by a factor of 10 using Ypet to replace EYFP as the new FRET receptor. The changes in the dynamics of the process by which vascular endothelial growth factor (VEGF) stimulated Src were first detected using this method.

1.5.3.3 FRET probes for other protease activity detection (1) Detection of secreted protease activity Matrix metalloproteinases (MMPs) are a category of enzymes with the ability to degrade the extracellular matrix and play an important role in growth, invasion, metastasis, and angiogenesis of malignant tumors. Generally, MMP is secreted into

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 Zhihong Zhang and Qingming Luo

the extracellular matrix in the form of inactive zymogen and then is subsequently activated after a series of enzyme digestion processes. Luo et al. artificially targeted the probe outside the cell membrane to detect the dynamics of extracellular MMP activation. This group joined a secreted peptide derived from Igκ to the N-terminus of MMP probe sequences based on CFP/YFP and simultaneously introduced transmembrane sequences to the C-terminus of the MMP probe, thereby positioning a fluorescent protein FRET probe on the outer surface of the cell membrane. The extracellular activated MMP can specifically cleave the MMP recognition sequence probe in the linker, which causes CFP and YFP to dissociate and FRET to disappear. (2) Detection of transmembrane protease activity β-Amyloid is abnormally produced and accumulated in the brains of Alzheimer’s disease (AD) patients and plays an important role in the pathogenesis of AD. After the precursor protein-amyloid precursor protein (APP) is sequentially cleaved by two proteases (β- and γ-secretase), Aβ is produced. Because the beta-site APP cleavage enzyme (BACE) is a key enzyme generated by Aβ and is closely related to the occurrence of AD, it is considered to be an important target for the treatment of AD. To detect BACE activity under high spatial and temporal resolution, Lu and Luo et al. used a CFP/YFP fluorescent protein and a BACE-specific substrate to construct the BACE-specific FRET probe. BACE activity in living cells has been successfully detected.

1.5.3.4 Parallel detection of multimolecular events based on FRET probes In general, many different molecules form a complex regulatory network that participates in a particular physiological or pathological event in cells. We can more closely examine the temporal characteristics and dynamic behavior of specific events in cells by combining optical imaging with a wide variety of fluorescent protein probes. The main challenge in application of multimolecular fluorescent protein probes is how best to rationally and efficiently allocate the limited visible spectrum (400–650 nm). Because fluorescent intensity ratio imaging is a quantitative method for the detection of FRET, the advantage of parallel detection of multimolecular events is that it will not be affected by probe concentration, photon bleaching, or other factors [174]. In ratio imaging, the signal spectra of the FRET probe fluorescent protein donor and acceptor must be collected simultaneously, and because fluorescent proteins have wide excitation and emission spectra, this makes the spectral distribution of such probes particularly important. In a single cell, we prefer to select FRET pairs without spectral cross talk to take advantage of fluorescent protein FRET probes and ratio imaging for detection of bimolecular events. In 2008, Piljic and Schultz et al. achieved synchronized imaging of four types of fluorescent probes in a single cell using the cytoplasm-localized



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­Calmodulin kinase probe CYCaMIIα (which is based on CFP/YFP), a membrane-localized PKC probe PM-CKAR (which is based on CFP/YFP), a cytoplasm-localized annexin A4 probe ORNEX4 and calcium probe (which are respectively based on mOrange/mCherry and Fura red (a chemical dye)). In this study, the researchers took advantage of the difference between the two fluorescent protein excitation spectra of CFP/YFP (405 nm) and mOrange/mCherry (561 nm), thereby avoiding spectral cross talk [158, 175]. To address the weakness of RFPs and far-RFPs as receptors, Ai et al. solved the problem of different spectral FRET probe pairs from another point of view. This group took advantage of the different excitation spectra of two FRET donors, that is, mTPF1, which was combined with YFP to form an FRET pair, and mAmetrine, which was combined with tdTomato to form an FRET pair. mTFP1 is excited using a 450 to 460-nm filter, whereas mAmetrine is excited using a 381 to 392-nm filter. The quantum yields of both acceptors of these two FRET pairs are high. The author designed two Caspase-3 probes based on mTPF1/YFP and mAmetrine/tdTomato and located them in the cytoplasm and nucleus, respectively. The two Caspase-3 probes accurately characterized Caspase-3 activation timing during apoptosis in the cell nucleus and cytoplasm. The results suggested that Caspase-3 in the cytoplasm was activated first. However, it should be noted that the emission spectra of the two probes are highly difficult to separate, resulting in cross talk. More specifically, 3 % of the spectrum of mAmetrine experiences cross talk with the mTPF/YFP FRET channel, and 14 % of the signal of mTPF1/YFP FRET displays cross talk with the mAmetrine channel. However, the author suggested that if the concentrations of the probes were similar, cross talk correction is not required [160]. In 2012, Su and Zhang et al. generated a new FRET pair via fluorescent protein spectral analysis. The donor and acceptor of the FRET pair were a YFP (mVenus) and an orange fluorescent protein (mKOκ), respectively. These researchers established a dual rate synchronous real-time imaging method by connecting an FRET pair based on mVenus/mKOκ (yellow/orange) with the single fluorescent protein probe Grx1-roGFP2 (green), thus achieving notably low interference ratio imaging and simultaneous real-time monitoring of the bimolecular events of Src signal and GSH redox potential within a single cell. Studies have shown that the Src signal induced by EGF could be negatively adjusted by H2O2, and this adjustment is achieved by H2O2 acting on the intracellular GSH redox system. In addition, this group also established a new method for dual rate synchronous real-time imaging based on an mVenus/ mKOκ (yellow/orange) FRET pair and an mTagBFP/sfGFP (blue/green) FRET pair, thus achieving notably low interference ratio imaging and simultaneous real-time monitoring of the bimolecular events of Src signal and Ca2+ signal within a single cytoplasm. As noted, development of additional new red-shift FRET probes is crucial for dynamic study of the relevance and timeliness of multiple molecular events in living cells [148, 153].

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Yu Li, Lingyu Zeng, Zhihong Liu, Jingui Qin

2 Two-photon Molecular Probe 2.1 Introduction of two-photon absorption In the early 20th century, the establishment of the quantum theories was one of the greatest events in physics and chemistry. Along with the establishment of these theories, people put forward a different basic unit, namely photons, from molecules or atoms. Through the interaction between photons and atoms or molecules, energy exchange happens between the radiation and the matter, which can be described as the absorption or emission of photons. In the early development of the quantum theory, scientists only considered the processes of either one-photon absorption or one-photon emission, both were called ­single-photon process. A one-photon process is that a molecule absorbs one photon from the incident light and simultaneously makes an energy transition from a lower energy level to a higher level, or on the contrary, the molecule emits one photon through a transition from a higher energy level to a lower level. Because of the conservation of energy, the photon energy must equal with the difference between the two energy levels. In 1931, the probability of two-photon absorption (2PA) was proposed by M. ­Göppert-Mayer [1] (1906–1972) in her doctoral dissertation for the first time, but this can be achieved only under the strong radiation [2, 3]. Because the 2PA effect of general materials is very weak, the research progress of 2PA was very slow until the advent of the laser. In the late 1980s, Parthenopoulos and Rentzepis [4] pioneered to study two-photon-induced photochromic reactions, and tried the application of the two-photon technology in 3D data storage. Then, Denk et al. [5] realized the potential prospect of application of the 2PA materials in fluorescence microscopic imaging, photodynamic therapy and medical diagnosis, and started the experiment exploration. In the midand late 1990s, the 2PA cross sections of some materials had reached several orders of magnitude higher than previously known materials, which triggered the increasing interest in the study of 2PA process [6, 7].

2.1.1 The basic concept of 2PA As shown in Figure 2.1, 2PA is a kind of third-order nonlinear optical phenomenon. Under the strong light excitation, molecules absorb two photons at the same time, Yu Li, Lingyu Zeng, Zhihong Liu (Corresponding Author) Jingui Qin (Corresponding Author): Department of Chemistry, Wuhan University, China, Email: [email protected]; [email protected] Yu Li, Lingyu Zeng: These authors contribute equally in this chapter. DOI 10.1515/9783110304596-002

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and are excited from the ground state to the excited state through an intermediate virtual state. The two photons either can be of the same frequency (degenerate TPA) or originate from two different frequencies. S2 S1

ω2

S2 SPA S0

ω1

TPA Fig. 2.1: Energy level diagrams for one-photon absorption transition and 2PA transition.

When the incident light is of a single frequency, we call it the degenerate 2PA process. In general cases, the incident light can originate from two different frequencies; the molecules can still absorb two photons with different frequencies at the same time and finish the transition between the two eigen energy levels, but must satisfy the law of conservation of energy. In this case, the process can be called the nondegenerate 2PA process. Two-photon absorption effect can be roughly described by means of nonlinear optical semiclassical theory. And it can also be described by the quantum electrodynamical theory with more rigorous and detailed instructions [8–10]. Nonlinear optical effect is that, under strong light excitation, the positive and negative charge centers of the molecules are migrated so as to show nonlinear polarization and other kinds of physical phenomena. In both macromedium (such as crystal, thin film) and micromedium (such as atom and molecule), an optical frequency electric polarization phenomenon can be triggered under a strong radiation. According to the nonlinear electric polarization effect of the semiclassical theory, the electric polarization vector P of a medium is the function of the light field-induced electric-dipole-moment vectors E, as shown below. (2.1) P = ε0[χ (1) E + χ (2) E 2 + χ (3) E3 ] Here, χ (1) is the first-order (linear) susceptibility of the medium; χ (2) is the second-order susceptibility; and χ (3) is the third-order susceptibility. Among them, χ (3) is a plural parameter corresponding to the third-order nonlinear optical effects: the real part does not involve the energy exchange between molecular systems and light field, and it can be used to describe the nonlinear refraction of medium depending on the light intensity; its imaginary part is associated with the nonlinear absorption of the material, which can determine the 2PA [10, 11]. The experimental results show that the numerical value, χ (1) > > χ (2) > > χ (3), and each level varies by about seven or eight orders of magnitude. Therefore, in the case of



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applying a weak light field from an incoherent light source, P is linearly proportional to the applied electric field E for common linear optical phenomenon; however, in the case of applying a strong light field, the polarization and photoelectric field intensity are nonlinearly co-related, and various nonlinear optical phenomenon can be observed, such as second harmonic generation, sum frequency, difference frequency and optical rectification of second-order nonlinear optical effects, or third-order nonlinear optical effects such as optical Kerr effect, four-wave mixing, three harmonic generation, and 2PA. A quasi-monochromatic, quasi-directional and strong beam of light passes through a nonlinear optical medium with an attenuation upon time, which can generally be expressed as [12]:

dI (z ) (2.2) = −αI (z ) − βI 2(z ) − γI 3(z ) − ηI 4(z )  dz Here, I(z) is the propagating intensity of the incident light beam along the z-axis, α, β, γ, and η are the one-, two-, three-, and four-photon absorption coefficients of the medium, respectively. If the medium is linearly transparent for the incident light, then there is no linear absorption (α = 0). At the case where only 2PA is considered, then the following equation applies:

dI (z ) (2.3) = −βI 2(z ) dz The physical meaning of the expression is that the probability of 2PA is proportional to the square of the local light intensity in a given position. The solution of the equation is:

I0(λ ) (2.4) I (z , λ ) = 1 + β(λ )I0(λ )z Here, I0(λ ) is the incident light intensity and β(λ ) is the 2PA coefficient (in units of cm/ GW), which depends on the wavelength of the incident light and is proportional to the molecular density (N0, in units of cm−3): (2.5) β(λ ) = δ2(λ )N0 = δ2(λ )NAc × 10−3 In which δ2 is the molecular 2PA cross sections (in units of cm4/GW), NA is Avogadro’s constant, and c is the molar concentration of the absorbing molecules (in units of mol/L).

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Different materials have different 2PA abilities; the strength of 2PA can be characterized by 2PA cross sections, which is only related to the molecular structure. In order to commemorate M. Göppert-Mayer, people adopt her name abbreviation now as the 2PA cross sections unit (1 GM ≡ 10−50 cm4 photons−1 molecule−1 s) [13]. At this point, the conversion relation of the 2PA cross sections δ2PA and the 2PA coefficient β is as follows:

hνβ × 103 (2.6) = hν × δ2 δ2PA = NAc

2.1.2 Measurements of 2PA effect Two-photon absorption cross section is generally more than 10 orders of magnitude lower than single-photon absorption cross section, and the test of 2PA is much more complex than that of single-photon absorption. There are many interference factors in the testing process, for example, the laser pulse width is one of the most important factors, and the 2PA cross sections of most compounds decrease with the shortness of laser pulse width, which is mainly related to excited-state absorption (ESA) [14]. The response of ESA is relatively slow, so the ESA can be effectively restrained with shorter pulse laser in the 2PA cross section test. Therefore, the two-photon test results using femtosecond laser are of higher reliability [15]. Two main techniques for measuring 2PA cross sections are the open z-scan technique and the two-photon excited fluorescence (TPEF). The other two less widely used techniques are nonlinear optical transmission [16, 17] and transient absorption, and these two techniques are beyond the scope of this paper. Next, we shall simply introduce the two commonly used techniques.

(1) The open z-scan technique In open z-scan technique, the sample pool is put on the laser channel, and is moved in the direction of the laser beam (z direction) around the focus (as shown in Figure 2.2). Because the incident light density on the sample is changing gradually, the nonlinear optical effect is different and the far field intensity distribution is changing, so as to give a function describing the relationship between light intensity and z-axis displacement [18]. If the detector only opens a small hole (the so-called “closed-aperture” setting), due to the third-order nonlinear polarization of the sample or heating effects, the light intensity change is sensitive to the refractive index, which leads to self-focusing or defocusing of the beam. On the contrary, if the detector collects all the light



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–z

–z0

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Computer

Sample Fig. 2.2: Experimental setup for the z-scan experiment.

of the sample (the so-called “open-aperture” settings), then the output only reflects the light transmission, and then the data can be used to calculate 2PA cross sections. Distinguishing feature of this method is that it is simple and highly sensitive, but the result is likely to be influenced by many factors leading to considerable experimental errors. Two main factors are: 1. If the hole of the detector is too small or too far from the sample pool, the light cannot be collected completely due to self-defocusing or nonlinear scattering, so that the measured value will be larger than the real one. 2. No matter the excitement is due to one-photon or two-photon absorption; the materials at excited state will further absorb incident light photons through ESA. The contribution from ESA can make the values of 2PA cross sections larger. The ESA can be reduced by the following three methods: the wavelengths used for sample are negligible for 1 PA, very short laser pulses (