By Wolfgang Becker
This publication is an try and bridge the distance among the instrumental ideas of multi-dimensional time-correlated unmarried photon counting (TCSPC) and ordinary purposes of the strategy. Written by way of an originator of the method and via sucessful clients, it covers the fundamental rules of the process, its interplay with optical imaging tools and its software to quite a lot of experimental projects in lifestyles sciences and medical research.
The ebook is usually recommended for all clients of time-resolved detection concepts in biology, bio-chemistry, spectroscopy of stay structures, stay mobile microscopy, scientific imaging, spectroscopy of unmarried molecules, and different purposes that require the detection of low-level gentle signs at single-photon sensitivity and picosecond time resolution.
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Extra info for Advanced Time-Correlated Single Photon Counting Applications
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17. Multi-wavelength FLIM is predominately used in applications where fluorescence signals of several fluorophores are present, and the emission of the fluorophores cannot cleanly separated by filters, or there are just too many fluorophores to get them separated by filters and dichroic beamsplitters. Multi-wavelength FLIM was first demonstrated in 2002 by Becker et al. for recording decay data in the complete donor-acceptor wavelength range of a FRET experiment [20]. Bird et al. demonstrated the technique for lifetime imaging of stained kidney tissue samples [28].
In applications were only a single optical waveform has to be recorded, or the experiment allows a series of waveforms to be recorded sequentially the classic principle is still used to a large extend. The typical application is recording of fluorescence decay curves. Examples are shown in Fig. 6. 6, left, shows fluorescence decay curves of quinine sulphate quenched by Cl− ions. The fluorescence lifetime changes with the quencher concentration [92]. 6, right shows decay curves of DODCI (3,3′diethyloxadicarbocyanine iodide) for different wavelength.
The colour shows the amplitude-weighted mean lifetime of a double-exponential decay. The image format is 512 × 512 pixels, every pixel contains 256 time channels. Decay curves in two selected pixels are shown on the right. Similar curves are contained in any pixel of the image. FLIM systems of the architecture shown in Fig. 22 are used for a wide range of applications. An overview is given in Chap. 3 of this book. The applications can essentially be divided into three classes: Measurement of parameters of the local molecular environment of the fluorophores (Chaps.
