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.     

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.     

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.     

Note: Optical trigger device with sub-picosecond timing jitter and stability

We are presenting the design, construction, and overall performance of the optical trigger device. This device generates an electrical signal synchronously to the detected ultra-short optical pulse. The device was designed for application in satellite laser ranging and laser time transfer experiments, time correlated photon counting and similar experiments, where picosecond timing resolution and detection delay stability are required. It consists of the ultrafast optical detector, signal discriminator, output pulse forming circuit, and output driver circuits. It was constructed as a single compact device to optimize their matching and maintain stability. The detector consists of an avalanche photodiode--both silicon and germanium types may be used to cover the wavelength range of 350-1550 nm. The analogue signal of this photodiode is sensed by the ultrafast comparator with 8 GHz bandwidth. The ps clock distribution circuit is used to generate the fast rise/fall time output pulses of pre-set length. The trigger device timing performance is excellent: the random component of the timing jitter is typically 880 fs, the temperature dependence of the detection delay was measured to be 370 fs/K. The systematic error contribution depends on the laser used and its stability. The sub-ps values have been obtained for various laser sources.     

Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two‐ to eight‐channel, time‐gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time‐gates are significantly more sensitive than the two time‐gate system. Only minor sensitivity differences were found between systems with four or more time‐gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP‐PMTs and 300–400 ps for other detectors. The maximum count rate of fast MCP‐PMTs, however, is 10–100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP‐PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.     

Software defined photon counting system for time resolved x-ray experiments

The time structure of synchrotron radiation allows time resolved experiments with sub-100 ps temporal resolution using a pump-probe approach. However, the relaxation time of the samples may require a lower repetition rate of the pump pulse compared to the full repetition rate of the x-ray pulses from the synchrotron. The use of only the x-ray pulse immediately following the pump pulse is not efficient and often requires special operation modes where only a few buckets of the storage ring are filled. We designed a novel software defined photon counting system that allows to implement a variety of pump-probe schemes at the full repetition rate. The high number of photon counters allows to detect the response of the sample at multiple time delays simultaneously, thus improving the efficiency of the experiment. The system has been successfully applied to time resolved scanning transmission x-ray microscopy. However, this technique is applicable more generally.     

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.     

Time-resolved soft x-ray absorption setup using multi-bunch operation modes at synchrotrons

Here, we report on a novel experimental apparatus for performing time-resolved soft x-ray absorption spectroscopy in the sub-ns time scale using non-hybrid multi-bunch mode synchrotron radiation. The present setup is based on a variable repetition rate Ti:sapphire laser (pump pulse) synchronized with the ~500 MHz x-ray synchrotron radiation bunches and on a detection system that discriminates and singles out the significant x-ray photon pulses by means of a custom made photon counting unit. The whole setup has been validated by measuring the time evolution of the L(3) absorption edge during the melting and the solidification of a Ge single crystal irradiated by an intense ultrafast laser pulse. These results pave the way for performing synchrotron time-resolved experiments in the sub-ns time domain with variable repetition rate exploiting the full flux of the synchrotron radiation.     

微脉冲激光雷达中的光子计数死区时间瞬态效应

建立了光子计数器的模型,对光子计数器的死区时间效应进行了理论分析和实际测量,并提出了校正光子计数效应对微脉冲雷达信号影响的方法。利用马尔科夫链对微脉冲雷达的探测过程进行理论分析,并利用MATLAB对死区时间对光子计数产生的影响进行了计算和分析,描述了计数死区对探测结果产生的瞬态形态变化。在此基础上,搭建了微脉冲激光雷达光子计数的测量平台,实验验证了死区时间对于光子计数采集的影响。最后,测量了不同光强下死区时间对探测结果的抑制情况,给出了微脉冲激光雷达信号的校正方法。基于提出的方法对真实微脉冲激光雷达信号进行了校正实验。实验结果表明:在峰值功率为100mW的405nm激光照射下,光子计数在采集频率100 MHz时的散射光计数效应减少了50%。文中的方法较好地解释了小尺寸目标的探测信号形态,实现了对光子计数探测结果的校正。     

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.     

Continuous measurement of the arrival times of x-ray photon sequence

In order to record x-ray pulse profile for x-ray pulsar-based navigation and timing, this paper presents a continuous, high-precision method for measuring arrival times of photon sequence with a common starting point. In this method, a high stability atomic clock is counted to measure the coarse time of arrival photon. A high resolution time-to-digital converter is used to measure the fine time of arrival photon. The coarse times and the fine times are recorded continuously and then transferred to computer memory by way of memory switch. The pulse profile is obtained by a special data processing method. A special circuit was developed and a low-level x-ray pulse profile measurement experiment system was setup. The arrival times of x-ray photon sequence can be consecutively recorded with a time resolution of 500 ps and the profile of x-ray pulse was constructed. The data also can be used for analysis by many other methods, such as statistical distribution of photon events per time interval, statistical distribution of time interval between two photon events, photon counting histogram, autocorrelation and higher order autocorrelation.     

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)).     

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.     

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.     

Self-suppression of reset induced triggering in picosecond SPAD timing circuits

We present a new photon timing circuit that achieves a time resolution of 35 ps full width at half maximum with single photon avalanche diodes having active area diameters up to 200 microm. The timing circuit is based on a double avalanche current sensing network that makes it particularly suited to operation at high photon counting rates. Thanks to its self-adjusting capabilities, no trimming is needed even when changing the photodetector operating conditions over a wide range.     

Holder for fast photodiodes in TO-18 package

The design of a holder for fast photodiodes is described. These holders have been tested with commercially available photodiodes in a standard TO-18 package using delta light pulses from a synchronously pumped cw dye laser. Results of the measurements are presented and the holder is shown to perform well even for an experimental photodiode with a rise time of less than 40 ps and a FWHM of 80 ps.     

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.     

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.     

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.     

Gamma bang time analysis at OMEGA

Absolute bang time measurements with the gas Cherenkov detector (GCD) and gamma reaction history (GRH) diagnostic have been performed to high precision at the OMEGA laser facility at the University of Rochester with bang time values for the two diagnostics agreeing to within 5 ps on average. X-ray timing measurements of laser-target coupling were used to calibrate a facility-generated laser timing fiducial with rms spreads in the measured coupling times of 9 ps for both GCD and GRH. Increased fusion yields at the National Ignition Facility (NIF) will allow for improved measurement precision with the GRH easily exceeding NIF system design requirements.