Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources

High brightness light emitting diodes are an inexpensive and versatile light source for wide‐field frequency‐domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio‐frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal‐to‐noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices (～40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal‐to‐noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.     

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.     

SLIM: a new method for molecular imaging

We used spectrally resolved fluorescence lifetime imaging (SLIM) to investigate the mitochondria staining dye rhodamine 123 and binding of DAPI to RNA and DNA in cells. Moreover, different components of the photosensitizer Photofrin were resolved in cell cultures by SLIM. To record lifetime images (tau-mapping) with spectral resolution we used a laser scanning microscope equipped with a spectrograph, a 16 channel multianode PMT, and multidimensional time-correlated single photon counting. A Ti:Saphir laser was used for excitation or alternatively a ps diode laser. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in cell cultures. As an example, the mitochondria staining dye rhodamine I23 could be easily distinguished from DAPI, which binds to nucleic acids. Also different binding sites of DAPI could be discriminated. This was proved by the appearance of different lifetime components within different spectral channels. Moreover, we were able to detect monomeric and aggregated forms of Photofrin in cells. Different lifetimes could be attributed to the various compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.     

Double-pulse fluorescence lifetime measurements

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique (Müller et al. , 1995, Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc 177, 171–179).
A laser source with an optical parametric amplifier (OPA) system is used to obtain short pulse durations needed for high time resolution, wavelength tunability for selective excitation of specific fluorophores and high pulse energies to obtain (partial) saturation of the optical transition.
It is shown that fluorescence lifetimes can be determined correctly also with nonuniform saturation conditions over the observation area.
A correction scheme for the effect on the measurements of laser power fluctuations, which are inherently present in OPA systems, is presented. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique.
Because signal detection does not require fast electronics, the technique can be readily used for fluorescence lifetime imaging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.     

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

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

Subnanosecond single photon counting fluorescence spectroscopy using synchronously pumped tunable dye laser excitation

A synchronously pumped tunable dye laser has been constructed and interfaced with a modified Ortec 9200 photon counting system for the purpose of measuring subnanosecond relaxation phenomena. The dye laser excitation pulse, which has an intrinsic 35-ps FWHM for Rhodamine 6G, is 350 ps when measured by time-correlated single photon counting. This value appears to be characteristic of the transit time jitter in the RCA 8850 photomultiplier tube. Subnanosecond fluorescence lifetimes of Rhodamine B with KI as a quencher have been determined by deconvolution of photons counted versus elapsed time using the method of moments; the shortest lifetime measured was 68 ps. Various technical aspects of the system are discussed with emphasis on applications to biophysical problems.     

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).     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Picosecond-resolution fluorescence lifetime measuring system with a cw laser and a radio

A procedure for measurement of fluorescence lifetimes with picosecond time resolution is described. A cw laser beam is modulated with a standing-wave acousto-optic modulator. The modulated beam is split; one part serves as a reference beam, the other part excites the fluorescent sample. The sample flourescence and the reference beam, attenuated and delayed optically to be equal in amplitude and opposite in phase to the fluorescence, are incident onto a single photomultiplier tube. The thus achieved photodetector ac null is monitored either by an AM radio, whose intermediate-frequency signal is displayed on an oscilloscope, or by a spectrum analyzer. With 30-MHz light modulation and the radio, lifetimes could be determined with resolution better than 15 ps. With the spectrum anlyzer and 170-MHz light modulation frequency we have achieved 4-ps lifetime resolution. Correction for photomultiplier transit time versus incident wavelength is made.     

Multispectral fluorescence lifetime imaging by TCSPC

We present a fluorescence lifetime imaging technique with simultaneous spectral and temporal resolution. The technique is fully compatible with the commonly used multiphoton microscopes and nondescanned (direct) detection. An image of the back-aperture of the microscope lens is projected on the input of a fiber bundle. The input of the fiber bundle is circular, and the output is flattened to match the input slit of a spectrograph. The spectrum at the output of the spectrograph is projected on a 16-anode PMT module. For each detected photon, the encoding logics of the PMT module deliver a timing pulse and the number of the PMT channel in which the photon was detected. The photons are accumulated by a multidimensional time-correlated single photon counting (TCSPC) process. The recording process builds up a four-dimensional photon distribution over the times of the photons in the excitation pulse period, the wavelengths of the photons, and the coordinates of the scan area. The method delivers a near-ideal counting efficiency and is capable of resolving double-exponential decay functions. We demonstrate the performance of the technique for autofluorescence imaging of tissue.     

Note: Fully integrated time-to-amplitude converter in Si-Ge technology

Over the past years an always growing interest has arisen about the measurement technique of time-correlated single photon counting TCSPC), since it allows the analysis of extremely fast and weak light waveforms with a picoseconds resolution. Consequently, many applications exploiting TCSPC have been developed in several fields such as medicine and chemistry. Moreover, the development of multianode PMT and of single photon avalanche diode arrays led to the realization of acquisition systems with several parallel channels to employ the TCSPC technique in even more applications. Since TCSPC basically consists of the measurement of the arrival time of a photon, the most important part of an acquisition chain is the time measurement block, which must have high resolution and low differential nonlinearity, and in order to realize multidimensional systems, it has to be integrated to reduce both cost and area. In this paper we present a fully integrated time-to-amplitude converter, built in 0.35?μm Si-Ge technology, characterized by a good time resolution (60 ps), low differential nonlinearity (better than 3% peak to peak), high counting rate (16 MHz), low and constant power dissipation (40 mW), and low area occupation (1.38×1.28?mm(2)).     

Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein

Time‐resolved microspectrofluorometry in live cells, based on time‐ and space‐correlated single‐photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal‐to‐noise ratio of 10⁶, and a spectral resolution of 30 (Δλ/Λ). As an exemple, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.     

Fluorescence lifetime imaging of lignin autofluorescence in normal and compression wood

Wood cell walls fluoresce as a result of UV and visible light excitation due to the presence of lignin. Fluorescence spectroscopy has revealed characteristic spectral differences in various wood types, notably normal and compression wood. In order to extend this method of characterising cell walls we examined the fluorescence lifetime of wood cell walls using TCSPC (Time‐Correlated Single Photon Counting) as a method of potentially detecting differences in lignin composition and measuring the molecular environment within cell walls. The fluorescence decay curves of both normal and compression wood from pine contain three exponential decay components with a mean lifetime of τ_m = 473 ps in normal wood and 418 ps in compression wood. Lifetimes are spatially resolved to different cell wall layers or cell types where individual lifetimes are shown to have a log‐normal distribution. The differences in fluorescence lifetime observed in pine compression wood compared to normal wood, are associated with known differences in cell wall composition such as increased p‐hydroxyphenyl content in lignin as well as novel deposition of β(1,4)‐Galactan. Our results indicate increased deposition of lignin fluorophores with shorter lifetimes in the outer secondary wall of compression wood. We have demonstrated the usefulness of fluorescence lifetime imaging for characterising wood cell walls, offering some advantages over conventional fluorescence imaging/spectroscopy. For example, we have measured significant changes in fluorescence lifetime resulting from changes to lignin composition as a result of compression wood formation that complement similar changes in fluorescence intensity.     

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.     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

Development and characterization of sub-100 ps photomultiplier tubes

We describe the evaluation of a microchannel plate (MCP) photomultiplier tube (PMT), incorporating a 3?μm pore MCP and constant voltage anode and cathode gaps. The use of the small pore size results in PMTs with response functions of the order of 85 ps full-width-half-maximum, while the constant electric field across the anode and cathode gaps produces a uniform response function over the entire operating range of the device. The PMT was characterized on a number of facilities and employed on gas Cherenkov detectors fielded on various deuterium tritium fuel (DT) implosions on the Omega Laser Facility at the University of Rochester. The Cherenkov detectors are part of diagnostic development to measure Gamma ray reaction history for DT implosions on the National Ignition Facility.     

Spatially resolved recording of transient fluorescence‐lifetime effects by line‐scanning TCSPC

We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one‐dimensional scan. The technique is based on scanning the sample with a high‐frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca²⁺ in cultured neurons. Microsc. Res. Tech. 77:216–224, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time‐domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.     

A 3D aligning method for stimulated emission depletion microscopy using fluorescence lifetime distribution

Optimal resolution by stimulated emission depletion (STED) microscopy requires precise alignment of the donut‐shaped depletion focus to the excitation focus. In this article, we demonstrate that fluorescence lifetime distribution can be implemented to align the STED system. Different from the traditional aligning methods in which a scattering imaging module is often equipped, the lifetime‐based method is free from probable mismatches between the scattering mode and the fluorescent mode, drift errors caused by separate imaging and complex fitting methods. Based on this method, a spatial resolution of 38 nm by time‐gated detection has been achieved. Microsc. Res. Tech. 77:935–940, 2014. © 2014 Wiley Periodicals, Inc.