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 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 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 system for in vivo studies

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.     

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 lifetime imaging microscopy: spatial resolution of biochemical processes in the cell

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.     

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 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 microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.     

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: 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.     

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 unravels C. trachomatis metabolism and its crosstalk with the host cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ₂-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ₂-NAD(P)H increase inside the chlamydial inclusion. τ₂-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ₂-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.     

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.     

Mesoscopic fluorescence lifetime imaging: Fundamental principles,clinical applications and future directions

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real-time assessments of tissue properties in clinical settings. Label-free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real-time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.     

Fluorescence lifetime: Beating the IRF and interpulse window

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes—outside the intermediate, Goldilocks, zone—for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.     

Fluorescence intensity decays of 2-aminopurine solutions: lifetime distribution approach

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.     

Fluorescence lifetime quenching and anisotropy studies of ribonuclease T1

The time-resolved fluorescence of the lone tryptophanyl residue of ribonuclease T1 was investigated by using a mode-locked, frequency-doubled picosecond dye laser. The fluorescence decay could be characterized by a single exponential function with a lifetime of 3.9 ns. The fluorescence was readily quenched by uncharged solutes but was unaffected by iodide ion. These observations are interpreted in terms of the electrostatic properties of the amino acid residues at the active site of the protein, which would appear to restrict the access of solute species to the tryptophanyl residue. The temperature dependence of the fluorescence lifetime and anisotropy decay time could be rationalized in terms of a model which postulates a significant ordering of the solvent layer immediately surrounding the surface of the protein.