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 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.     

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.     

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.     

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.     

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.     

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.     

Operation of silicon single photon avalanche diodes at cryogenic temperature

This article reports a complete characterization of single photon avalanche diodes (SPADs) at temperatures down to 120 K. We show that deep cooling of the device by means of a compact liquid-nitrogen Dewar brings several advantages, such as extremely low dark counting rates (down to 1 counts/s), better time resolution, and higher quantum efficiency in the visible range. By using a special current pick-off circuit, we achieved a time resolution of 20 ps full width at half maximum at 120 K for a 50 mum diameter SPAD. Afterpulsing effects are avoided by using a sufficiently long hold-off time (microseconds).     

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.     

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.     

High-linearity analog-to-digital acquisition board for photon-timing applications

Nowadays a wide range of scientific applications require the detection of very weak and fast luminescence signals in the time domain. Single molecule spectroscopy, diffuse optical tomography, time-resolved emission spectra are only a few examples among them. Advanced time-correlated single photon counting, that relies on multidetector and multidimensional acquisition, reveals itself as a powerful technique to gather deeper insight into various photochemical and biological processes. In this paper we present a high-speed, high-linearity A/D acquisition board that is designed as a building block for a compact, low-cost, multidetector acquisition system for photon-timing experiments. The prototype has been experimentally tested and the results fully comply the goal design specifications.     

High-energy x-ray backlighter spectrum measurements using calibrated image plates

The x-ray spectrum between 18 and 88 keV generated by a petawatt laser driven x-ray backlighter target was measured using a 12-channel differential filter pair spectrometer. The spectrometer consists of a series of filter pairs on a Ta mask coupled with an x-ray sensitive image plate. A calibration of Fuji? MS and SR image plates was conducted using a tungsten anode x-ray source and the resulting calibration applied to the design of the Ross pair spectrometer. Additionally, the fade rate and resolution of the image plate system were measured for quantitative radiographic applications. The conversion efficiency of laser energy into silver Kα x rays from a petawatt laser target was measured using the differential filter pair spectrometer and compared to measurements using a single photon counting charge coupled device.     

用单光子计数法测量稀土荧光材料的激发光谱

单光子计数法是弱光测量领域中的一种新技术,在生物、化学和医学等领域中具有十分重要的作用。本文介绍了新设计的稀土荧光材料激发光谱测量系统,从信号电平指出了普通模拟测量方法所存在的问题。详细讨论了设计的单光子计数测量系统。通过几个样品的测量结果表明,单光子计数法是荧光光谱等极微弱光测量中十分有效的手段     

Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels

Time-correlated single photon counting is a powerful method for sensitive time-resolved fluorescence measurements down to the single molecule level. The method is based on the precisely timed registration of single photons of a fluorescence signal. Historically, its primary goal was the determination of fluorescence lifetimes upon optical excitation by a short light pulse. This goal is still important today and therefore has a strong influence on instrument design. However, modifications and extensions of the early designs allow for the recovery of much more information from the detected photons and enable entirely new applications. Here, we present a new instrument that captures single photon events on multiple synchronized channels with picosecond resolution and over virtually unlimited time spans. This is achieved by means of crystal-locked time digitizers with high resolution and very short dead time. Subsequent event processing in programmable logic permits classical histogramming as well as time tagging of individual photons and their streaming to the host computer. Through the latter, any algorithms and methods for the analysis of fluorescence dynamics can be implemented either in real time or offline. Instrument test results from single molecule applications will be presented.     

用于激光测距的高精度时间数字转换电路

针对大容量现场可编程门阵列(FPGA)时间数字转换电路线性度较差的问题,采用小容量FPGA实现了用于激光测距的高精度、高线性度时间数字转换电路。通过对高速计数器、数字插入方法、编码器硬件算法的研究,分析了影响时间数字转换电路精度和非线性误差的因素,提出了一种降低非线性误差的方法。首先,根据所分析的影响因素,解决了高速锁存的问题,在单片小容量FGPA XC2V250上实现了时间数字转换电路;接着,通过USB接口将携带时间信息的计数器值和温度计码转为二进制编码值传给PC机,进行计算和显示;最后,设计了延时测量电路,对所设计的时间数字转换电路进行了测试,得到了各个延时单元延时的大小,并进行了数据分析和处理。测试结果显示:时间数字转换电路单次测时分辨率约为80 ps,校正后可达40 ps左右,微分非线性误差为-0.524LSB~+0.448LSB,积分非线性误差为-1.598LSB~+1.492LSB,可以满足飞行时间法激光测距中高精度测时的要求。     

Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub-diffraction particles with high spatial and temporal resolutions

In photon event distribution sampling, an image formation technique for scanning microscopes, the maximum likelihood position of origin of each detected photon is acquired as a data set rather than binning photons in pixels. Subsequently, an intensity-related probability density function describing the uncertainty associated with the photon position measurement is applied to each position and individual photon intensity distributions are summed to form an image. Compared to pixel-based images, photon event distribution sampling images exhibit increased signal-to-noise and comparable spatial resolution. Photon event distribution sampling is superior to pixel-based image formation in recognizing the presence of structured (non-random) photon distributions at low photon counts and permits use of non-raster scanning patterns. A photon event distribution sampling based method for localizing single particles derived from a multi-variate normal distribution is more precise than statistical (Gaussian) fitting to pixel-based images. Using the multi-variate normal distribution method, non-raster scanning and a typical confocal microscope, localizations with 8 nm precision were achieved at 10 ms sampling rates with acquisition of ~200 photons per frame. Single nanometre precision was obtained with a greater number of photons per frame. In summary, photon event distribution sampling provides an efficient way to form images when low numbers of photons are involved and permits particle tracking with confocal point-scanning microscopes with nanometre precision deep within specimens.     

单光子荧光时间谱仪时间分辨率的设计和测定

探讨了影响时间分辨单元光子荧光谱仪时间分辨率的因素,组装和调试了三种类型的时间相关单光子荧光谱仪,测定了它们的激发光脉冲响应函数,提出了测定时间分辨率的方法,并测定了时间分辨率。     

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.     

A novel fluorescence lifetime imaging system that optimizes photon efficiency

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

Integration of close range photogrammetry and expert system capabilities in order to design and implement optical image based measurement systems for intelligent diagnosing disease

In medical applications and disease diagnosis devices, image is considered as a tool for measurement and data acquisition. Most of the imaging methods usually used in medical applications are invasive and have several side effects on human body. So, other types of image based measurement systems should be developed for medical applications. The systems must be able to use the images captured in visible part of the electromagnetic spectrum. In this research a new image based disease diagnosis method has been developed which uses optical images for measuring required symptoms. In the systems which are implanted based on the suggested method measurement capabilities of close range photogrammetry and decision making ability of expert system are integrated. The integrated system can be used for the diseases whose symptoms are visible or appear as deformations out of body and around the affected area. For evaluation of the suggested method, an integrated system has been designed and implemented for intelligent diagnosing foot deformity.