Fluorescence detection with high time resolution: from optical microscopy to simultaneous force and fluorescence spectroscopy

Picosecond time-resolution fluorescence signal detection over many hours is possible using the time-correlated single photon counting (TCSPC) technique. Advanced TCSPC with clock oscillator set by the pulsed laser and data analysis provides a tool to investigate processes in single molecules on time scale from picoseconds to seconds. Optical imaging techniques combined with TCSPC allow one to study the spatial distribution of fluorescence properties in solution and on a surface. Mechanical manipulation of a single macromolecule by means of an atomic-force microscope makes it possible to detect fluorescence signal changes as a function of mechanical conformations of a fluorescent dye attached to a single DNA molecule.     

Fluorescence lifetime imaging by time-correlated single-photon counting

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.     

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.     

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)).     

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.     

Single Photon Counting With Synchronously Pumped Dye Laser Excitation

ABSTRACT

This article reviews the advances that have been made in the technique of pulse fluorometry with time-correlated single photon counting detection brought about by the introduction of mode-locked synchronously pumped dye laser excitation. High repetition rates and small pulse width permit high data collection rates and excellent time-resolution. A modern pulse fluorometer which allows efficient measurement of fluorescence decay curves as well as automated measurement of time-resolved fluorescence spectra and of fluorescence anisotropy decays is described in detail.     

Two-photon lifetime imaging of fluorescent probes in intact blood vessels: a window to sub-cellular structural information and binding status

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.     

单分散上转换纳米荧光微粒的荧光寿命测量

为了表征上转换纳米荧光微粒的发光特性,设计了一个可以对单个纳米微粒进行荧光寿命测量的系统。该系统首先使用基于检流计振镜的双光子显微镜系统对单分散状态的上转换纳米微粒样品进行扫描成像。然后,通过单分子荧光纳米定位算法精确找出每个纳米微粒的准确位置,再依次将激光聚焦到每个纳米微粒上,在该点施加一个500μs宽度的激光脉冲,并通过光电倍增管探测随时间变化的荧光强度信号。最后对荧光衰减曲线进行拟合,计算得到该纳米微粒的荧光寿命。实验结果表明:单个上转换纳米荧光微粒的荧光发射曲线符合单指数衰减规律,其荧光寿命为195.3μs。与之相比,聚集状态的纳米微粒的荧光寿命为358.9μs。这表明聚集状态对上转换纳米微粒的发光特性有显著影响。     

Subnanosecond time-correlated photon counting with tunable lasers

We present several laser based methods to improve the technique of time-correlated photon counting. Our Ar(+) laser pumped tunable dye laser can be operated in three timing configurations: acousto-optically mode locked, cavity dumped, and cavity dumped-mode locked. Performance characteristics of the laser system in various operational modes are described along with measurement techniques for both gas and liquid phase. The subnanosecond pulses generated by mode locking are extremely stable and they maintain identical pulse shapes over a 6-h period, as shown via photon counting measurements at a 15-psec channel resolution. Our RCA C31034 photomultiplier with a red sensitive GaAs photocathode provides wavelength-independent response to detected fluorescence in both the visible and ultraviolet. The present limit of our apparatus is controlled by the accuracy of deconvoluting fluorescence decay from the finite response width caused by photomultiplier transit time dispersion (0.8 nsec FWHM). Our system stability is sufficient to accurately determine exponential decays as short as 50 psec. Furthermore, we can successfully analyze dual exponential decays such as those arising from solution reorientation times of 390 psec competing with a fluorescence lifetime of 725 psec. Examples of the laser performance are selected from a variety of measurements in the gas phase and from the fluorescent dye rose bengal in the liquid phase.     

Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source

We report the demonstration of time‐correlated single‐photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white‐light supercontinuum excitation source. By using a 100 fs‐pulsed Ti:Sapphire laser with a low‐frequency chirp to pump a 30‐cm long section of photonic crystal fibre, a ps‐pulsed white‐light supercontinuum was created. Optical bandpass interference filters were then applied to this broad‐bandwidth source to select the 488‐nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non‐infected material. The white‐light generation source provides a flexible method of producing variable‐bandwidth visible and ps‐pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.     

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.     

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting.     

Towards metabolic mapping of the human retina

Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.     

AlGaAs DH pump laser for photoluminescence lifetime measurements

The use of GaAlAs double heterostructure lasers as a pulsed excitation source for photoluminescence time-decay measurements is described. Subnanosecond laser pulses easily allow the determination of luminescence decay times >/=500 ps using a single photon counting system. In contrast to mode-locked gas or dye lasers, this new technique utilizes simple equipment (diode laser and pulse generator) and requires no special alignment or tuning procedures.     

A multi-view time-domain non-contact diffuse optical tomography scanner with dual wavelength detection for intrinsic and fluorescence small animal imaging

We present a non-contact diffuse optical tomography (DOT) scanner with multi-view detection (over 360°) for localizing fluorescent markers in scattering and absorbing media, in particular small animals. It relies on time-domain detection after short pulse laser excitation. Ultrafast time-correlated single photon counting and photomultiplier tubes are used for time-domain measurements. For light collection, seven free-space optics non-contact dual wavelength detection channels comprising 14 detectors overall are placed around the subject, allowing the measurement of time point-spread functions at both excitation and fluorescence wavelengths. The scanner is endowed with a stereo camera pair for measuring the outer shape of the subject in 3D. Surface and DOT measurements are acquired simultaneously with the same laser beam. The hardware and software architecture of the scanner are discussed. Phantoms are used to validate the instrument. Results on the localization of fluorescent point-like inclusions immersed in a scattering and absorbing object are presented. The localization algorithm relies on distance ranging based on the measurement of early photons arrival times at different positions around the subject. This requires exquisite timing accuracy from the scanner. Further exploiting this capability, we show results on the effect of a scattering hetereogenity on the arrival time of early photons. These results demonstrate that our scanner provides all that is necessary for reconstructing images of small animals using full tomographic reconstruction algorithms, which will be the next step. Through its free-space optics design and the short pulse laser used, our scanner shows unprecedented timing resolution compared to other multi-view time-domain scanners.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

High-performance time-resolved fluorescence by direct waveform recording

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1?μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats.     

Optimized time‐gated generalized polarization imaging of Laurdan and di‐4‐ANEPPDHQ for membrane order image contrast enhancement

The membrane dyes Laurdan and di‐4‐ANEPPDHQ can be used to image membrane order due to a spectral blue‐shift in the fluorescence emission between the liquid‐ordered and liquid‐disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, we exploit the known excited state photophysics and time‐resolved data acquisition via time‐correlated single‐photon counting (TCSPC) to demonstrate GP contrast enhancement for these two probes of 7 and 31%, respectively. This improvement in image contrast enhancement will be invaluable when studying the role of lipid rafts in fixed and live cell systems. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for "double kinetics" experiments: Application to fluorescence resonance excitation energy transfer study of protein folding

The information obtained by studying fluorescence decay of labeled biopolymers is a major resource for understanding the dynamics of their conformations and interactions. The lifetime of the excited states of probes attached to macromolecules is in the nanosecond time regime, and hence, a series of snapshot decay curves of such probes might - in principle - yield details of fast changes of ensembles of labeled molecules down to sub-microsecond time resolution. Hence, a major current challenge is the development of instruments for the low noise detection of fluorescence decay curves within the shortest possible time intervals. Here, we report the development of an instrument, picosecond double kinetics apparatus, that enables recording of multiple fluorescence decay curves with picosecond excitation pulses over wide spectral range during microsecond data collection for each curve. The design is based on recording and averaging multiphoton pulses of fluorescence decay using a fast 13 GHz oscilloscope during microsecond time intervals at selected time points over the course of a chemical reaction or conformational transition. We tested this instrument in a double kinetics experiment using reference probes (N-acetyl-tryptophanamide). Very low stochastic noise level was attained, and reliable multi-parameter analysis such as derivation of distance distributions from time resolved FRET (fluorescence resonance excitation energy transfer) measurements was achieved. The advantage of the pulse recording and averaging approach used here relative to double kinetics methods based on the established time correlated single photon counting method, is that in the pulse recording approach, averaging of substantially fewer kinetic experiments is sufficient for obtaining the data. This results in a major reduction in the consumption of labeled samples, which in many cases, enables the performance of important experiments that were not previously feasible.     

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.