A novel fluorescence lifetime imaging system that optimizes photon efficiency

Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination.     

A practical implementation of multifrequency widefield frequency‐domain fluorescence lifetime imaging microscopy

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Herein, we describe a practical implementation of multifrequency widefield FD‐FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine‐wave modulation. This allows parallel multifrequency FLIM measurement using the Fast Fourier Transform and the cross‐correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restores the loss of optical resolution caused by the defocusing effect when the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit‐free lifetime analysis of FLIM images. Here, our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multifrequency FLIM system is a valuable and simple tool in fluorescence imaging studies. Microsc. Res. Tech. 76:282–289, 2013. © 2013 Wiley Periodicals, Inc.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Multiple frequency fluorescence lifetime imaging microscopy

The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set‐up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto‐optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons ‘on’ and ‘off’ as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the ‘on’ state of the intensifier relative to its ‘off’ state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square‐pulse modulation. A phase‐dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel‐by‐pixel basis using a non‐linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co‐expressed in live cells.     

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.     

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly- l -lysine and poly- d -lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d -isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.     

Image analysis for denoising full-field frequency-domain fluorescence lifetime images

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).     

φFLIM: a new method to avoid aliasing in frequency-domain fluorescence lifetime imaging microscopy

In conventional wide‐field frequency‐domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity‐modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, φFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360° range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide‐field frequency‐domain FLIM system based on an acousto‐optic modulator for modulation of the excitation and a microchannel‐plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to ± 29° and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP‐transfected HeLa cells were as high as 100%. With φFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.     

Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient

Fluorescence lifetime‐resolved images of chlorophyll fluorescence were acquired at the maximum P‐level and during the slower transient (up to 250 s, including P‐S‐M‐T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P‐level, wild type and the violaxanthin‐accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime‐resolved images. The zeaxanthin‐accumulating mutant npq2 displays reduced fluorescence intensity at the P‐level (about 25–35% less) and corresponding lifetime‐resolved frequency domain phase and modulation values compared to wild type/npq1. A two‐component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a ‘dimmer switch’. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime‐resolved imaging measurements for cells that were treated with the electron transport inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P‐level and during the slow fluorescence transient, compared to wild type and the npq1 mutant. The fluorescence lifetime‐resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The above results are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so‐called ‘State Transitions’, and on non‐photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two ‘lifetime pools’ centred on slower and faster fluorescence lifetimes.     

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.     

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.     

Measurement of nanosecond time-resolved fluorescence with a directly gated interline CCD camera

CCD cameras coupled optically to gated image intensifiers have been used for fast time‐resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the ‘charge drain’ function on an interline‐transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.     

Low-cost, frequency-domain, fluorescence lifetime confocal microscopy

We describe the theory and implementation of a frequency‐domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications‐type, radio‐frequency electronics are used to provide inexpensive modulation references and to perform phase‐sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.     

Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation

The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to approximately 2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to approximately 2.0 ns when stored within secretory vesicles.     

Digital scanned laser light-sheet fluorescence lifetime microscopy with wide-field time-gated imaging

We develop a multidimensional fluorescence imaging technique by implementing a wide-field time-gated fluorescence lifetime imaging into digital scanned laser light-sheet microscopy (FLIM-DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time-gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM-DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.     

Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM)

Membrane type 1 matrix metalloproteinase (MT1‐MMP) is a membrane‐tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1‐MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1‐MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT‐1080 were imaged using an intracellular Förster resonance energy transfer‐based biosensor specific for MT1‐MMP; a substrate specific for MT1‐MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer‐based sensor. The configuration of the biosensor was determined with fluorescence lifetime‐resolved imaging microscopy using both a polar plot‐based analysis and a rapid data acquisition modality of fluorescence lifetime‐resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1‐MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1‐MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.     

Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data

Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error‐weighting results in a non‐linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime‐based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.     

Multidie LED combined with homogenizing optics to improve frequency response and intensity for FLIM applications

Application of light‐emitting diodes (LEDs) in frequency‐domain fluorescence lifetime imaging microscopy (FLIM) has been limited by the trade‐off between modulation frequency and illumination intensity of LEDs, which affects the signal‐to‐noise ratio in fluorescence lifetime measurements. To increase modulation frequency without sacrificing output power of LEDs, we propose to use LEDs with multiple dice connected in series. The LED capacitance was reduced with series connection; therefore, the frequency response of multidie LED was significantly increased. LEDs in visible light, including blue, green, amber and red, were all applicable in FLIM. We also present a homogenizing optics design, so that multidie LEDs produced uniform illumination on the same focal spot. When the homogenizing optics was combined with multicolour emitters, it provides multiple colour selection in a compact and convenient design.     

Scanning-less wide-field single-photon counting device for fluorescence intensity, lifetime and time-resolved anisotropy imaging microscopy

A scanning‐less single‐photon counting system for FLIM and fluorescence anisotropy wide‐field imaging is described and characterized in this paper. The two polarizations of the fluorescence are divided by a Glan prism and acquired at the same time by the Q_A detector. Fluorescence decay profiles can be reconstructed for any desired area up to each pixel and used to calculate time‐resolved fluorescence anisotropy decays.     

A modified phasor approach for analyzing time-gated fluorescence lifetime images

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (～10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.