Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

We present a novel, multi‐dimensional, time‐correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser‐scanning microscope operated at a pixel dwell‐time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi‐exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed‐distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non‐FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1‐43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4‐64, an efficient energy acceptor for FM1‐43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high‐resolution two‐dimensional images of lifetime distributions in living neurones.     

Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells

Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.     

Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells

In this work, we characterized streptavidin-conjugated quantum dots (QDs) manufactured by Quantum Dot Corporation. We present data on: (1) two-photon excitation; (2) fluorescence lifetimes; (3) ensemble and single QD emission anisotropy; (4) QDs as donors for Forster resonance energy transfer (FRET); and (5) spectral conversion of QDs exposed to high-intensity illumination. We also demonstrate the utility of QDs for (1) imaging the binding and uptake of biotinylated transferrin on living cells, and (2) resolving by fluorescence lifetime imaging microscopy (FLIM) signals originating from QDs from those of spatially and spectrally overlapping visible fluorescent proteins (VFPs).     

Multi-dimensional fluorescence lifetime and FRET measurements

When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of F?rster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.     

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc.     

Imaging FRET standards by steady-state fluorescence and lifetime methods

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.     

Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.     

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)‐fusion proteins co‐expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N‐myristoylation motif of the calcium‐dependent protein kinase 1 (LeCPK1) of tomato. Upon co‐expressing in cowpea protoplasts a perfect co‐localization at the plasma membrane of the constructs was observed. Acceptor‐photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co‐expressing CFP‐Zm7hvr and myrLeCPK1‐YFP, whereas no FRET was apparent in protoplasts co‐expressing CFP‐Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP‐Zm7hvr and myrLeCPK1‐YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP‐Zm7hvr was reduced in the presence of myrLeCPK1‐YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor‐bleached protoplast co‐expressing CFP‐Zm7hvr and myrLeCPK1‐YFP revealed slow requenching of the CFP fluorescence in the acceptor‐bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1‐YFP in the complex with CFP‐Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.     

Photoswitchable cyan fluorescent protein as a FRET donor

Among a variety of fluorescent proteins available today, there is a lack of suitable markers with excitation/emission in the violet/blue part of visible spectrum. Recently, we reported on photoswitchable cyan fluorescent protein (PS-CFP), which represents monomeric high-contrasting photactivatable label for in vivo protein movement tracking. However, PS-CFP demands high intensity of light for the photoswitching. Therefore it can be employed as a common fluorescent tag at conventional light intensities, which cause negligible or zero photoactivation. High pH stability and unique positioning of excitation/emission peaks make it a worthy supplement to the existing palette of fluorescent proteins. Here we use PS-CFP fusion with a yellow fluorescent protein phiYFP to show that PS-CFP is a promising donor partner for the fluorescence resonance energy transfer (FRET). A remarkable phenomenon is that PS-CFP donor fluorescence turned to be essentially stable with and without FRET, while acceptor emission demonstrated record dynamic range of up to 7.8-fold. This makes the FRET pair presented a useful tool for the single color high throughput screenings. Here we also propose ways for further PS-CFP enhancing, aiming to develop bright cyan fluorescent protein with unique spectral characteristics.     

Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor‐only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (R_C) of acceptor‐to‐donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission‐spectral unmixing FRET methodology, Iem‐spFRET, which can simultaneously measure the E and R_C of FRET sample without any external references, such that Iem‐spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem‐spFRET by measuring the absolute E and R_C values of standard constructs with different acceptor‐to‐donor stoichiometry expressed in living cells. Our results demonstrate that Iem‐spFRET is a simple and powerful tool for real‐time monitoring the dynamic intermolecular interaction within single living cells.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

Combining ocFLIM and FIDSAM reveals fast and dynamic physiological responses at subcellular resolution in living plant cells

For a deeper understanding of molecular mechanisms within cells and for the realization of predictive biology for intracellular processes at subcellular level, quantitative biology is required. Therefore, novel optical and spectroscopic technologies with quantitative and dynamic output are needed in cell biology. Here, we present a combined approach of novel one-chromophore fluorescence lifetime imaging microscopy to probe the local environment of fluorescent fusion proteins and fluorescence intensity decay shape analysis microscopy to suppress interfering autofluorescence. By applying these techniques, we are able to analyse the subcellular localization and partitioning of a green fluorescence protein fusion of the salt stress-induced protein low temperature induced (LTI)6b in great detail with high spatial and temporal resolution in living cells of Arabidopsis plants.     

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

A spectrograph with continuous wavelength resolution has been integrated into a frequency‐domain fluorescence lifetime‐resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross‐talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral‐FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral‐FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime – and thereby the FRET efficiency – without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.     

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching

To study protein–protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor–acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein–protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.     

FRET measurements on fuzzy fluorescent nanostructures

In the last decade, fluorescence resonance energy transfer (FRET) has become a useful technique for studying intermolecular interactions applied to the analysis of biological systems. Although FRET measurements may be very helpful in the comprehension of different cellular processes, it can be difficult to obtain quantitative results, hence the necessity of studying FRET on controllable systems. Here, a fuzzy nanostructured system called a nanocapsule is presented as a nanometric-device allowing distance modulation, thus preserving photophysical properties of fluorescent dyes and exhibiting good potential features for improving quantitative FRET analysis. We evaluated the behavior of such a sample using four FRET methods (three of them based on steady-state fluorescence and one using lifetime measurements). Within some limitations that can be overcome, these nanodevices have the potential to serve as a benchmark system for characterizing new FRET couples and to develop quantitative approaches for FRET analysis.     

Fluorescence lifetime imaging--techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time‐domain FLIM by multidimensional time‐correlated single photon counting, time‐domain FLIM by gated image intensifiers, frequency‐domain FLIM by gain‐modulated image intensifiers, and frequency‐domain FLIM by gain‐modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein‐interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity‐based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM‐FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM‐based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM‐FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady‐state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady‐state fluorescence techniques.     

Towards metabolic mapping of the human retina

Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.     

Applying spectral fingerprinting to the analysis of FRET images

F?rster resonance energy transfer (FRET) allows one to study interactions between two fluorescently labeled molecules (donors and acceptors) at distances on the order of 5 nm. Many studies have described methods of how to measure the efficiency of FRET. However, few have addressed the question of how fluorescence from unpaired donors and acceptors can be determined in addition to that from FRET-pairs and how the signal-to-noise ratio (SNR) of such estimates depends on the presence of the partner species. Such knowledge, however, is essential for many biological applications, in which-after initial characterization of the spectral properties of a well-defined donor-acceptor complex-the in vivo affinity and stoichiometry of the complex is of interest. Here, we provide a theoretical analysis on how spectral fingerprinting can be applied to separate fluorescence of FRET pairs from that originating from unpaired donors and acceptors and how to select imaging parameters to optimize the SNR of the estimates. Thereby, we uncover a fundamental problem in this application and discuss ways to evade its adverse consequences. We compare the expected resolution of traditional FRET measures with that of optimized spectral fingerprinting and analyze the resolution of a method for FRET measurements that combines spectral with fluorescence lifetime information.