Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells

Quantitative analysis in Förster resonance energy transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two-component system (FRET and no FRET donor species), fitting of fluorescence lifetime imaging microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (f_D) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mf_D), coming from the mathematical minimization of f_D. We find particular advantage in the use of mf_D because it can be obtained without fitting procedures and it is derived directly from FLIM data. mf_D constitutes an interesting quantitative parameter for live cell studies because it is related to the minimal relative concentration of interacting proteins. For multi-lifetime donors, the process of fitting complex fluorescence decays to find at least four reliable lifetimes is a near impossible task. Here, mf_D extension for multi-lifetime donors is the only quantitative determinant. We applied this methodology for imaging the interaction between the bromodomains of TAF_II250 and acetylated histones H4 in living cells at high resolution. We show the existence of discrete acetylated chromatin domains where the minimal fraction of bromodomain interacting with acetylated H4 oscillates from 0.26 to 0.36 and whose size is smaller than half of one micron cube. We demonstrate that mf_D by itself is a useful tool to investigate quantitatively protein interactions in live cells, especially when using fast FRET-FLIM acquisition times.     

Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f_D) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f_D percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f_D value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f_D (mf_D) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f_D analysis.     

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).     

High-precision FLIM-FRET in fixed and living cells reveals heterogeneity in a simple CFP-YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor-acceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements.     

FRET-FLIM applications in plant systems

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.     

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.     

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mf_D. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels N_ch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, N_ch or small fractions of interacting donor f_D). The validity domain of the moments method is more restricted (not applicable when f_D<0.25 or when N_ch = 4) but it is more precise than the polar approach. We also demonstrate that the mf_D is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.     

Imaging protein-protein interactions in living cells

The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules. Methods to visualize interaction can be based on FRET (fluorescence detected resonance energy transfer), for example in fluorescence lifetime imaging microscopy (FLIM). Another method is based on fluorescence correlation spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein interactions in vivo are reviewed.     

FLIM-FRET analyzer: open source software for automation of lifetime-based FRET analysis

Background

Despite the broad use of FRET techniques, available methods for analyzing protein-protein interaction are subject to high labor and lack of systematic analysis. We propose an open source software allowing the quantitative analysis of fluorescence lifetime imaging (FLIM) while integrating the steady-state fluorescence intensity information for protein-protein interaction studies.

Findings

Our developed open source software is dedicated to fluorescence lifetime imaging microscopy (FLIM) data obtained from Becker & Hickl SPC-830. FLIM-FRET analyzer includes: a user-friendly interface enabling automated intensity-based segmentation into single cells, time-resolved fluorescence data fitting to lifetime value for each segmented objects, batch capability, and data representation with donor lifetime versus acceptor/donor intensity quantification as a measure of protein-protein interactions.

Conclusions

The FLIM-FRET analyzer software is a flexible application for lifetime-based FRET analysis. The application, the C#. NET source code, and detailed documentation are freely available at the following URL: http://FLIM-analyzer.ip-korea.org.

     

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo^1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest^3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells⁵, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium⁶, cAMP^7-8, inositol phosphates⁹ and cGMP^10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.     

Fluorescence Lifetime Imaging of Interactions between Golgi Tethering Factors and Small GTPases in Plants

Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta . Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1^b, AtRab-H1^c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

FRET microscopy in the living cell: different approaches, strengths and weaknesses

New imaging methodologies in quantitative fluorescence microscopy, such as F?rster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.     

Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Background

Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k_D→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.

Methodology/Principal Findings

The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.

Conclusions/Significance

We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.     

Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: The case of Euglena gracilis

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A_498, non fluorescent and B₄₆₂, fluorescent.Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A₄₉₈ becomes B₄₆₂), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B₄₆₂ form acts as donor for the non-fluorescent A₄₉₈ form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.     

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis: PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES*

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)³ in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.     

Protein localization in living cells and tissues using FRET and FLIM

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.     

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor''s fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor''s emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005). Consequently, conventional imaging of GFP and its closest spectral variants (like cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP]) is most likely to be problematic in roots, whereas red-shifted intrinsic fluorescent proteins (including monomeric red fluorescent protein and recently identified spectral variants like mStrawberry and mCherry) may be hard to discriminate in chloroplast-containing aerial tissues (Chapman et al., 2005). The problems get further compounded in FRET assays because the autofluorescence arising from phenols, lignin, and chlorophyll can limit the choice of fluorophores suitable for in planta FRET assays.CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006; Dixit et al., 2006). However, in photophysical terms, this pair is less than ideal for FRET imaging. Both have broad excitation and emission spectra with a small Stokes shift (Chapman et al., 2005). Second, QY of CFP (QY = 0.4) is relatively lower than that of YFP (QY = 0.61), and thus a significantly higher (and rather cell damaging) amount of excitation energy is needed to induce FRET (Dixit et al., 2006). Additionally, CFP displays multiexponential lifetimes with a shorter (1.3 ns) and a longer (2.6 ns) component (Becker et al., 2006). Although the deviation from the single-component decay is reasonably small (Tramier et al., 2002; Becker et al., 2006), the shorter CFP lifetime component can erroneously be interpreted as being the result of lifetime reduction due to energy transfer. At the same time, weak or transient protein associations may get masked and thus remain undetected. Whereas the parental wild-type GFP is extremely photostable and shows a monoexponential decay pattern (excited-state lifetime 3.16 ± 0.03 ns; Striker et al., 1999; Volkmer et al., 2000; Shaner et al., 2005), its close spectral overlap with YFP makes it unsuitable as a donor in GFP-YFP FRET experiments. Likewise, wild-type or enhanced GFP (or YFP) as a donor to red-shifted monomers as acceptors is suboptimal because the 488-nm (or 514-nm) laser line commonly used to excite GFP (or YFP) cross excites most of the red monomers (e.g. mOrange, mStrawberry) because of their broad excitation spectra (Zapata-Hommer and Griesbeck, 2003; Shaner et al., 2004).Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003). The T203I mutation in TSapphire (and original Sapphire as well) abolishes the 475-nm excitation peak found in the wild-type GFP (Tsien, 1998). TSapphire is efficiently excited below 410 nm, which makes it ideal for studying plant protein dynamics and interactions because, at this wavelength, there is negligible excitation of the autofluorescing chlorophyll pigments. Furthermore, TSapphire also represents a good donor to red monomer acceptors that are negligibly excited at this wavelength (Shaner et al., 2004). Using a purified Zn²⁺ sensor with TSapphire and mOrange as a donor-acceptor pair, Shaner and colleagues demonstrated the ratiometric intramolecular FRET between the two fluorophores in vitro (Shaner et al., 2004). The sensor yielded a 6-fold ratiometric increase (562/514-nm mOrange/TSapphire emission ratio) upon Zn²⁺ binding.However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999; Volkmer et al., 2000). Furthermore, we demonstrate intramolecular FRET-FLIM between chimerically linked TSapphire and mOrange (with a 16-amino acid linker in between). When fused to genetically known interacting proteins and expressed in intact living cells, the donor and the acceptor fluorophores show energy transfer in different subcellular compartments indicative of intermolecular protein-protein interactions. These results validate the versatility of the proposed in vivo FRET-FLIM assay based on the donor TSapphire and the acceptor mOrange, which turns out to work with both soluble and membrane proteins.     

Spectral Unmixing: Analysis of Performance in the Olfactory Bulb In Vivo

Background

The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.

Methodology/Principal Findings

Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180–250 µm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.

Conclusions/Significance

Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.