Advanced Time-Correlated Single Photon Counting Applications by Wolfgang Becker

By Wolfgang Becker

This ebook is an try to bridge the space among the instrumental ideas of multi-dimensional time-correlated unmarried photon counting (TCSPC) and general purposes of the procedure. Written by way of an originator of the approach and via sucessful clients, it covers the elemental rules of the strategy, its interplay with optical imaging equipment and its program to quite a lot of experimental projects in existence sciences and medical research.

The ebook is suggested for all clients of time-resolved detection innovations in biology, bio-chemistry, spectroscopy of dwell platforms, stay mobile microscopy, scientific imaging, spectroscopy of unmarried molecules, and different functions that require the detection of low-level mild indications at single-photon sensitivity and picosecond time resolution.

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The authors found changes in the fluorescence spectra and shorter fluorescence lifetime in malignant melanoma compared to normal skin. 3 FLIM with Excitation Wavelength Multiplexing FLIM can be combined with excitation wavelength multiplexing. The general principle of multiplexed TCSPC is shown in Fig. 16. The extension of the principle to FLIM is shown in Fig. 29. Excitation at different wavelength is achieved by multiplexing (on/off switching) of several lasers, or by switching the wavelength of the acousto-optical filter (AOTF) of a super-continuum laser.

E. by shifting a time-gate over the signal, would record a distorted waveform in these cases. As a serious drawback of TCSPC it is often stated that the technique can record only one photon per signal period. If the light intensity is high a possible second or third photon in the same excitation pulse period with the first one is lost. The result is a distortion of the recorded signal waveform. Pile-up was indeed a serious problem in early TCPC experiments working at pulse repetition rates on the order of 10 kHz.

3, Fig. 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].

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