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.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.     

Time-domain fluorescence lifetime imaging applied to biological tissue.

Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue autofluorescence, it reveals intrinsic excellent contrast between different types and states of tissue. This article aims to review our recent progress in developing time-domain FLIM technology for microscopy and endoscopy and applying it to biological tissue.     

Fluorescence lifetime imaging to detect actomyosin states in mammalian muscle sarcomeres

We investigated the use of fluorescence lifetime imaging microscopy (FLIM) of a fluorescently labeled ATP analog (3'-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP) to probe in permeabilized muscle fibers the changes in the environment of the nucleotide binding pocket caused by interaction with actin. Spatial averaging of FLIM data of muscle sarcomeres reduces photon noise, permitting detailed analysis of the fluorescence decay profiles. FLIM reveals that the lifetime of the nucleotide, in its ADP form because of the low concentration of nucleotide present, changes depending on whether the nucleotide is free in solution or bound to myosin, and on whether the myosin is bound to actin in an actomyosin complex. Characterization of the fluorescence decays by a multiexponential function allowed us to resolve the lifetimes and amplitudes of each of these populations, namely, the fluorophore bound to myosin, bound to actin, in an actomyosin complex, and free in the filament lattice. This novel application of FLIM to muscle fibers shows that with spatial averaging, detailed information about the nature of nucleotide complexes can be derived.     

A flexible wide‐field FLIM endoscope utilising blue excitation light for label‐free contrast of tissue

Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide‐field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ～mm‐scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ～8 seconds using an average excitation power of ～0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode‐locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F?rster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.     

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

Background

Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.

Methodology/Principal Findings

We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.

Conclusions/Significance

NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.     

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and space-correlated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10mW/cm²). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.     

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein‐protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero‐FRET and homo‐FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Fluorescence lifetime imaging of calcium using Quin-2.

We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.     

Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.     

Hypoxia induces free radical damage to rat erythrocytes and spleen: analysis of the fluorescent end-products of lipid peroxidation.

Several studies have shown that hypoxia induces alterations in the lipid membranes of many cell types. The mechanism of these changes might consist in membrane lipid peroxidation. Lipid peroxidation in erythrocytes and spleen is easily detected by measurement of the concentration of fluorescent end-products. Exposure of rats to hypoxia for various time periods induced formation of lipophilic fluorescent products both in erythrocytes and spleen. A new kind of fluorophore was found in chloroform extracts from erythrocytes with excitation maximum at 270 nm and emission maximum at 310 nm. Additionally, two minor fluorophores were observed, emitting at 360 nm and in the region of 415-440 nm. Only one type of fluorophore was detected in spleen, emitting at 445 nm after excitation at 315 nm. The concentration of fluorophores was dependent on the time of hypoxic exposure both in erythrocytes and spleen. In erythrocytes there was a decrease of the predominant fluorophore after 3 hours (54%, P < 0.05) and 21 days (54%, P < 0.05) of hypoxia in relation to normoxic controls, accompanied by changes in spectral patterns of tridimensional fluorescence spectra. There was also a significant increase in the concentration of fluorophore in spleen (to 164%, P < 0.05, after 3 h, and to 240%, P < 0.05, after 21 days). The fluorophores, both in erythrocytes and spleen, were resolved into several distinct fractions with HPLC. The presented results support the hypothesis of hypoxia-induced lipid peroxidation and create a basis for further characterization of the fluorescent products.     

Development of a molecular bioswitch using fluorescence lifetime imaging: Incremental activation of fluorescein diacetate

Molecular bioswitches offer an invaluable asset in the shift from systemic to targeted treatments. Within the growing arsenal of switches are imaging probes that functionalize only in given locations or situations. Acetate esters are a common fluorescent example, known to activate upon interaction with esterases. Fluorescein diacetate (FDA) is one such fluorophore used in cell viability assays. These assays rely on the fact that the compound begins colorless and with no fluorescent signature whatsoever, and only after internalization into cells it is possible to detect a fluorescence signal. In this study, using fluorescence intensity (FI) and fluorescence lifetime (FLT) imaging, FDA is shown to be fluorescent even when unactivated. Furthermore, the FLT is shown to change with pH. Finally, the ability to image FDA in different environments simulated by tissue‐imitating phantoms is explored. Altogether, the ability of FDA to serve as a bioswitch when measured using FLT imaging microscopy (FLIM) is assessed. The combination of a spectrum of FDA activation and FLIM serves as a bioswitch, where biologically relevant stimulation can generate detectable and incremental variations.      

State transitions and photosystems spatially resolved in individual cells of the cyanobacterium Synechococcus elongatus

State transitions are a low-light acclimation response through which the excitation of Photosystem I (PSI) and Photosystem II (PSII) is balanced; however, our understanding of this process in cyanobacteria remains poor. Here, picosecond fluorescence kinetics was recorded for the cyanobacterium Synechococcus elongatus using fluorescence lifetime imaging microscopy (FLIM), both upon chlorophyll a and phycobilisome (PBS) excitation. Fluorescence kinetics of single cells obtained using FLIM were compared with those of ensembles of cells obtained with time-resolved fluorescence spectroscopy. The global distribution of PSI and PSII and PBSs was mapped making use of their fluorescence kinetics. Both radial and lateral heterogeneity were found in the distribution of the photosystems. State transitions were studied at the level of single cells. FLIM results show that PSII quenching occurs in all cells, irrespective of their state (I or II). In S. elongatus cells, this quenching is enhanced in State II. Furthermore, the decrease of PSII fluorescence in State II was homogeneous throughout the cells, despite the inhomogeneous PSI/PSII ratio. Finally, some disconnected PBSs were resolved in most State II cells. Taken together our data show that PSI is enriched in the inner thylakoid, while state transitions occur homogeneously throughout the cell.

During state transitions, the ratio of quenched and unquenched photosystem II complexes is homogeneously changed in individual cells of the cyanobacterium Synechococcus elongatus.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Non fitting based FRET–FLIM analysis approaches applied to quantify protein–protein interactions in live cells

New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET–FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET–FLIM to still be quantitative.     

Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm²) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm²) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.