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Time-Correlated Single Photon Counting Modules SPC-130EMN, SPC-130EMNX, SPC-130IN, SPC-130INX, SPC-150N, SPC-150NX, SPC-150NXX, SPC-160, SPC-160PCIE, SPC-180N, SPC-180NX, SPC-180NXX Detectors, Lasers and Peripheral Devices Simple-Tau Systems Technical Principles TCSPC Applications FLIM Systems Applications in Life Sciences Clinical FLIM Applications SPCM Software SPCImage NG Data Analysis Software Time-correlated single photon counting (TCSPC) is an amazingly sensitive technique for recording low-level light signals with picosecond resolution and extremely high precision.TCSPC originates from the measurement of excited nuclear states and has been used since the late 60s [775, 1250]. For many years TCSPC was used primarily to record fluorescence decay curves of organic dyes in solution. Due to the low intensity and low repetition rate of the light sources and the limited speed of the electronics of the 70s and 80s the acquisition times were extremely long. More important, classic TCSPC was intrinsically one-dimensional, i.e. limited to the recording of the waveform of a periodic light signal. Light sources ceased to be a limitation when the first mode-locked Argon lasers and synchronously pumped dye lasers were introduced. For the recording electronics, the situation changed with the introduction of the SPC-300 modules of Becker & Hickl in 1993. Due to a new analog-to-digital conversion principle these modules could be used at photon count rates almost 100 times higher than the classic TCSPC devices. Moreover, the modules were able to record the photons of a large number of detectors simultaneously. They were thus able to record a photon distribution not only versus the time in a fluorescence decay but also versus aspatial coordinate or the wavelength of the photons. Multi-dimensional TCSPC was born. Within a few years, more dimensions were added to multidimensional TCSPC. Fast sequential recording was introduced with the SPC-430 in 1995, fast scanning with the SPC-535 in 1997. Time-tag recording was introduced with the SPC-431 in 1996; multi-module TCSPC systems followed in 1999. Since then, the Becker & Hickl TCSPC systems became bigger, faster and more flexible. Recent TCSPC modules, like the SPC-150NX or the SPC-180, can be configured for sequential recording, imaging, or time-tag recording by a simple software command. Multi-module systems, like the SPC-134EM and SPC-154, can be used for scanning at unprecedented count rates and acquisition speeds. Nevertheless, TCSPC still has the reputation to be an extremely sluggish technique unable to record any fast changes in the fluorescence or scattering behaviour of a sample. The multidimensional features of modern TCSPC are not commonly understood. Thus, many users do not make efficient use of their SPC modules. However, if appropriately used, multidimensional TCSPC techniques not only deliver superior results but also solve highly sophisticated measurement problems. This handbook is an attempt to help existing and potential users understand and make use of the advanced features of modern TCSPC. After an introduction into the bh TCSPC devices and associated detector, laser, and experiment control modules the principles of advanced TCSPC techniques are described. These include multidetector TCSPC, multiplexed TCSPC, sequential recording techniques, scanning techniques, parameter-tag recording, and multi-module TCSPC techniques. The next chapter describes the architecture of the bh SPC modules. A chapter about detectors gives a review of detector principles and of the parameters used to characterise detectors. It describes a number of detectors commonly used for TCSPC and gives advice about obtaining best performance from them. The implementation of bh SPC devices is described in the next part of the handbook. It includes principles and wiring diagrams for typical experiments, guidelines for first system setup, and advice for system optimisation. It describes dead-time, counting loss, and pile-up effects, detector effects, and effects related to the optical system. The next chapter of the handbook is dedicated to TCSPC applications. The first part of this chapter describes the measurement of fluorescence and anisotropy decay curves, multispectral lifetime experiments, recording of transient fluorescence lifetime phenomena, and measurements of phosphorescence decay curves. The second part of the chapter is dedicated to time-resolved laser scanning microscopy. It contains sections on a wide variety of fluorescence-lifetime imaging (FLIM) experiments and procedures, such as FLIM with various excitation principles, excitation sources, and detection principles, high-speed and time-series FLIM, Z-stack FLIM, simultaneous fluorescence and phosphorescence lifetime imaging (FLIM/PLIM), fluorescence lifetime-transient scanning (FLITS), and FLIM with special microscope configurations. A third part contains FLIM background knowledge: Signal-to-noise ratio, acquisition time, the effect of counting loss and pile-up, photobleaching, and fluorescence depolarisation on the recorded data. The book contains a large chapter on TCSPC applications, most of them in Biology. It contains sections on FLIM of molecular environment parameters in tissue, FLIM-based FRET measurements in cells, autofluorescence FLIM of biological tissue, plant physiology, and clinical FLIM applications. A section about diffuse optical tomography (DOT) by NIRS techniques includes breast imaging, static and functional brain imaging, perfusion measurement in the human brain, diffuse tissue spectroscopy, and small-animal imaging. Picosecond photon correlation, fluorescence correlation spectroscopy, burst-integrated fluorescence lifetime techniques, and photon counting histogram techniques are reviewed in the next sections. The last part of the application chapter gives an review of non-biological TCSPC applications like positron lifetime measurement, measurement of barrier discharges, remote sensing, metrological applications, and characterisation of detectors. The application chapter also includes practical hints about optical systems, detectors, and other technical aspects of the applications described. Another large chapter describes the SPCM operating software of the bh SPC modules. It describes the various user interface configurations, operation modes, the system and control parameters, the handling and display of the multidimensional data recorded by the modules, and the associated data file structure. The TCSPC Handbook also contains a chapter on the SPCImage NG fluorescence decay and FLIM data analysis software. It describes the general principles of fluorescence decay analysis, the calculation of fluorescence decay parameters and lifetime images by various decay models, pseudo-global analysis, multi-wavelength FLIM analysis, batch-processing of FLIM series, and analysis of PLIM data. The handbook ends with a list of more than 1200 references related to TCSPC, most of them being applications of the bh SPC devices.
