TCSPC laser scanning microscope

Fig. 5.76 TCSPC laser scanning microscope with nondescanned (direct) detection...

Fig. 5.114 FLIM image (left), decay curve (middle) and FCS curve (right) obtained in a TCSPC laser scanning microscope. [42] and courtesy of Zdenek Petrasek and Petra Schwille, Biotec TU Dresden...

Dual-wavelength TCSPC detection in two-photon laser scanning microscopes is relatively simple [37]. Multispectral TCSPC detection in a two-photon laser scanning microscope requires a suitable relay optics between the objective lens and the polychromator [35, 60]. Details are described under TCSPC Laser Scanning Microscopy . 

An application of TCSPC FLIM to CFP-YFP FRET is shown in Fig. 5.87 and Fig. 5.88. The microscope was a Zeiss LSM 510 NLO two-photon laser scanning microscope in the Axiovert 200 version. An excitation wavelength of 860 nm was used. The nondescanned fluorescence signal from the sample was fed out of the... 

In the Scan Sync In mode, the scanner controls the TCSPC module. The scan controller is programmed to scan the image with a defined number of pixels per line and lines per frame. The scanner delivers a frame pulse when the scanning starts, a line pulse at the beginning of each line, and a pixel pulse at the transition to the next pixel within a line. The recording then runs in the same way as described for the laser scanning microscope with fast beam scanning. 

Side-window PMTs are rugged and inexpensive, and they often have a somewhat higher cathode efficiency than front window PMTs. Their broad TTS and long SER pulses make them less useful for TCSPC applications. However, side-window PMTs are used in many fluorescence spectrometers, in femtosecond correlators, and in laser scanning microscopes. If instruments like these have to be upgraded with TCSPC, it can be difficult to replace the detector. Therefore, some typical instrument response functions for side-window PMTs are given below. 

At present dedicated TCSPC FLIM boards are commercially available. They are compatible with most LSMS and are easily synchronized with the scanning microscope and pulsed laser. These boards, often plug-in cards for PCs, have a lower deadtime than do the conventional TCSPC electronics intended for use in spectroscopy and the memory bottle neck of the histogram-ming memory has been removed [21, 22], Consequently, these dedicated boards provide higher acquisition speeds. 

Leica has developed a microscope with a fast descanned FLIM detector connected directly to the scan head. The instrument uses a Becker Hickl SPC-830 TCSPC module and is available with a Ti Sapphire laser ( MP FLIM ) or a pulsed... 

The benefit of TCSPC wide-field imaging is that it can be easily adapted to almost any microscope or other optical system. It may also be a solution for samples that preclude, for whatever reason, scanning by a laser spot of high power density. 

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