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.     

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.     

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.     

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.     

Recent Developments in Frequency-Domain Fluorometry

I. SUMMARY

Phase-modulation fluorometry is the frequency-domain analogue of time-resolved fluorescence spectroscopy. During the past three years we witnessed the development of variable-frequency phase-modulation fluorometers with modulation frequencies from 1 to 220 MHz. These instruments provide impressive resolution of multi or non-exponential fluorescence decays. To introduce these instruments we describe their design and operational principles. To illustrate the obtainable resolution we present results for the resolution of two and three-component mixtures of fluorophores, the resolution of complex anisotropy decays from non-spherical molecules and the determination of time-resolved emission spectra in the presence of time-dependent spectral relaxation. At present, the resolution obtainable with the frequency-domain fluorometers appears to be at least equivalent to that obtained with pulsed mode-locked laser sources with single photon counting. These mode-locked sources can also be used for phase fluorometry, as described elsewhere in this volume by Gratton and co-workers.     

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。     

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 images and correlation spectra obtained by multidimensional time-correlated single photon counting

Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.     

An efficient spectroscopic detection system for cathodoluminescence mode scanning electron microscopy (SEM)

A detection system for analytical cathodoluminescence (CL) mode scanning electron microscopy (SEM) is described. This incorporates a cold stage, an efficient light collector, a monochromator and a photomultiplier and used the photon counting technique. The efficiency of the component subsystems was optimized and calibrated, and the performance of alternative light collecting and monochromating equipment is compared. The operation of the photon counter is discussed. The digital output of the photon counter was fed into a multichannel scaler and thence to a computer. This was used to correct the observed count rate with the calibrated spectral variations in the performance of the detection system. Spectra obtained at both room temperature and liquid nitrogen temperacure and monochromatic and panchromatic SEM micrographs are given as examples illustrating the value of this technique. The factors governing the performance of the system are discussed. The forms of noise in the signal and in the detection system are described and the means for minimizing, avoiding or correcting for them are dealt with. Sources of spurious signals in the SEM are treated.     

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.     

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.     

Tracking polymer diffusion in a wet latex film with fluorescence resonance energy transfer

We describe an instrument to measure the polymer interdiffusion between donor-labeled and acceptor-labeled latex polymers in a partially wet latex film with fluorescence resonance energy transfer (FRET). It is possible to temporarily arrest the drying process of a wet latex film by sealing the film in an airtight chamber. In our approach, we measure donor fluorescence decays from 0.5 mm diameter spots at various positions across an arrested latex film with time-correlated single photon counting. We interpret the resulting decays with a Monte Carlo simulation of the FRET process and extract information about the extent of polymer diffusion as a function of position on the film. These results enable us to determine the extent of polymer interdiffusion as a function of distance from the wet-dry edge in the latex film. To highlight this device's ability to capture the rapid early stages of latex interdiffusion, we report results from an acrylate copolymer latex.     

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.     

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.     

Automated luminescent topographic system

A new optical-scanning technique with a spatial resolution of 10 mum has been developed for the observation of surface luminescence. The photoluminescence topographic technique is a fully automated system capable of measuring luminescence intensity from a surface area as large as 25.8 cm(3) as well as the actual luminescence spectra from a spot as small as 10 mum in diameter. The system consists of an Ar-ion laser for excitation, a bidirectional scanner with focusing lens, a spectrometer for high spectral resolution, and a photomultiplier for photon counting. Experimental control and data acquisition and display are performed by a Hewlett-Packard 9820A calculator and interface package. Applications of the technique in the analysis of impurity segregation in semiconductor wafers are illustrated.     

Multiphoton fluorescence lifetime imaging of human hair

In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.     

A Multifrequency Phase Fluorometer Using the Harmonic Content of a Mode-Locked Laser

ABSTRACT

We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source.

A mode-locked argon ion laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repetition frequency of the laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz. Use of cross-correlation techniques permits precise isolation of individual frequencies. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using coupled frequency synthesizers. In the present instrument three synthesizers are used. One drives the pump mode-locker head, a second drives the acousto-optic modulator and the third is used to modulate the response of the photomultiplier tubes which detect the signal. The accuracy, reproducibility and sensitivity of the instrumentation have been determined. Experimental data are provided to show use of this high frequency cross-correlation phase-modulation fluorometer for the determination of fluorescence lifetimes and rotational motions of tryptophan in solution and in proteins.     

Picosecond Fluorescence Spectroscopy By Time-Correlated Single-Photon Counting

Abstract:

We describe the recent progress made in our laboratory on the time-resolved fluorescence spectroscopy by the combination of stable short-duration pulses from a CW mode-locked laser and a time-correlated single-photon counting method. Particular emphasis is placed on the achievement of high time resolution with side-on and microchannel-plate photomultipliers. We also discuss the limitation of using this method in the picosecond time range. Several applications of this spectroscopy such as the measurements of fluorescence decay characteristics and time behavior of fluorescence depolarization are reported. It is also shown that the high sensitivity of this method with pico-second time resolution is very suitable for the temporal rejection of cumbersome background-fluorescence in Raman spectroscopy. Finally, we present a novel method of photon time-of-flight fluorescence spectroscopy, in which an optical fiber is employed as a dispersive element.     

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.