Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

Fluorescence lifetime imaging microscopy in the medical sciences

The steady improvement in the imaging of cellular processes in living tissue over the last 10–15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).     

Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

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.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

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.     

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.     

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM).

BACKGROUND: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. METHODS AND RESULTS: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simulate, display, and discuss the effects of blurring and fluorophore heterogeneity on lifetime imaging in widefield and confocal configurations. CONCLUSION: Sectioning improves the quality of lifetime images of samples with multiple fluorophores or spatially varying F?rster resonance energy transfer.     

Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells.

A new method of fluorescence microscopy for cell imaging has been developed that takes advantage of the spatial variations of fluorescence lifetimes in single cells as a source of image contrast, and thus it is named "fluorescence lifetime imaging microscopy (flimscopy)". Since time-resolved fluorescence measurements are sensitive to molecular dynamics and interactions, flimscopy allows the molecular information to be visualized in single cells. In flimscopy measurements, several (nanosecond) time-resolved fluorescence images of a sample are obtained at various delay times after pulsed laser excitation of the microscope's entire field of view. Lifetimes are calculated pixel-by-pixel from these time-resolved images, and the spatial variations of the lifetimes are then displayed in a pseudocolor format (flimscopy image). The total data acquisition time needed to obtain a flimscopy image with the diffraction-limited spatial resolution (approximately 250 nm) is decreased to just approximately 30 s for approximately 300 fluorescent molecules/micron2. This was achieved by developing a high-frequency (400 kHz) nanosecond-gating (9 ns full width at half height)-signal accumulation system. This technique allows the extent of resonance energy transfer to be visualized in single living cells, and is free from the errors due to variations in path length, light scattering, and the number of fluorophores that necessitate complex corrections in steady-state microfluorometry and fluorescence ratio imaging microscopy. Flimscopy was applied here to observe the extent of fusion of individual endosomes in single cells. Results revealed the occurrence of extensive fusion between primary endocytic vesicles and/or sorting endosomes, thereby raising the possibility that the biogenesis of sorting endosomes involves multiple fusions of primary endocytic vesicles.     

Fluorescence lifetime and quenching studies on some interesting diphenylhexatriene membrane probes

The fluorescence lifetimes of a number of membrane probes based on the 1,6-diphenylhexatriene (DPH) chromophore have been measured in small unilamellar phospholipid vesicles and found to be multiphasic. These probes were quenched by sodium iodide with different efficiencies in vesicles and this has been attributed to the depth of the particular probe in the bilayer. The distribution of the probe between the outer and inner monolayer has been determined for those probes with fixed positions in the bilayer. The iodide ion permeability of the bilayer was found to be immeasurably small over a 3 h period.     

Fluorescence lifetime and anisotropy studies with liver alcohol dehydrogenase and its complexes

From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer

BACKGROUND: DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. METHODS: We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). RESULTS: The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. CONCLUSIONS: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.     

Fluorescence lifetime and polarization anisotropy studies of membrane surfaces with pyridoxal 5'-phosphate

The fluorescence lifetime and depolarization of the pyridoxal 5'-phosphate label demonstrated different environments at the structure-solvent interface for micelles, liposomes, proteins and membranes. A short lifetime and rotational correlation time for the micelles and liposomes proved that the label was strongly associated with the water solvent and rotated freely about the covalent bond. The proteins provided a more buried or hydrophobic site as shown by an increase in the lifetimes. Rotational correlation times of 4-6 ns for sarcolemma and erythrocyte membranes suggested restricted rotation for the pyridoxal 5'-phosphate label. Lower values of the rotational correlation time for rod outer segment and myelin sheath proved that the protein epsilon-amino groups are at the solvent interface which allows for more rotation.     

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.     

Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis

Fluorescence lifetime imaging microscopy presents a powerful tool in biology and biophysics because it allows the investigation of the local environment of a fluorochrome in living cells in a quantitative manner. Furthermore, imaging F?rster-type resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy enables protein-protein interactions and intermolecular distances to be mapped under physiological conditions. Quantitative and precise data analysis methods are required to access the richness of information that is contained in FRET data on biological samples. Lifetime detection in the frequency-domain yields two lifetime estimations. The lifetime moments analysis (LiMA) provides a quantitative measure of the lifetime distribution broadness by exploiting the analytical relationship between the phase- and demodulation-lifetime estimations and relating them to the weighted average and variance of the lifetime distribution. The LiMA theoretical framework is validated by comparison with global analysis and by applying it to a constrained two-component FRET system using simulations and experiments. Furthermore, a novel LIMA-based error analysis and a more intuitive formalism for global analysis are presented. Finally, a new method to resolve a FRET system is proposed and experimentally applied to the investigation of protein-protein interactions.     

Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-beta-cyclodextrin.     

Fluorescence lifetime imaging: association of cortical actin with a PIP3-rich membrane compartment

We have used fluorescence lifetime imaging (FLIM) to study actin and plasma membrane dynamics in B16-F1 melanoma cells. In the absence of a FRET acceptor, significant changes in the fluorescence lifetime of GFP were induced simply by linking the fluorophore to different functional probes, including beta-actin, the PH domains of PLCdelta and Akt, the Ras farnesylation signal, and the neuromodulin palmitoylation signal (MEM). In contrast, the lifetime of GFP-actin was constant despite the many different local environments of G- and F-actin within the cell. Treatment with cytochalasin D but not latrunculin A significantly shortened the lifetime of GFP-beta-actin in the absence of a FRET acceptor. Robust lifetime shifts were observed using either a GFP-RFP chimera or co-transfection of GFP-MEM with RFP-MEM. In contrast to previous reports we observed a photobleaching-dependent change in the lifetime of GFP which could complicate the interpretation of FRET experiments. Of the membrane probes tested only the fluorescence lifetime of GFP-Akt was influenced by the presence of mRFP-actin, suggesting that the cortical actin meshwork is associated with a PIP3-enriched compartment of the plasma membrane. These results will aid in the design of new FRET-based approaches to study cytoskeletal interactions at the molecular level.