TCSPC Laser Scanning Microscopy

However, the fluorescence of organic molecules is not only characterised by the intensity and the emission spectram, it also has a characteristic lifetime. The lifetime can be used as an additional parameter to separate the emission of different fluorophores, to probe ion concentrations and binding states in cells, and to investigate interactions between proteins by fluorescence resonance energy transfer. 

The application of the lifetime as a separation parameter is particularly useful to distinguish the autofluorescence components in tissues. These components often have poorly defined fluorescence spectra but can be distinguished by their fluorescence lifetime [282, 339, 517]. FLIM has also been used to verify the laser-based transfection of cells with GFP [501]. 

The distance between two different fiuorophore molecules can be probed by fluorescence resonance energy transfer (FRET) [308]. The energy transfer rate from the donor to the aeeeptor depends on the sixth power of the distance. FRET becomes noticeable at distanees on the order of a few mn and therefore occurs only if the donor and aeeeptor are physically linked. With FLIM techniques, FRET results are obtained from a single lifetime image of the donor [15, 32, 38, 61, 62, 63, 73, 80, 93, 147, 209, 405, 508]. 

The fluorescence lifetimes of typical fluorophores used in cell imaging are of the order of a few ns. However, the lifetime of autofiuorescence components and of the quenched donor fraetion in FRET experiments can be as short as 100 ps. In cells, lifetimes of dye aggregates as short as 50 ps have been found [261]. The lifetime of fluorophores eonneeted to metallic nanoparticles [182, 183, 309, 337] can be 100 ps and shorter. 

The local environment, the binding or aggregation state, the quenching rate, and the FRET efficiency of the fiuorophore moleeules in cells are normally inhomogeneous. Moreover, different fluorophores may overlap within the same pixel. Therefore, the fluorescenee deeay funetions found in cells are usually multiexponential. A FLIM technique should not only resolve lifetimes down to 50 ps, it should also be able to resolve multiexponential deeay funetions. 

---

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 . 

Technical Details of TCSPC Laser Scanning Microscopy... 

The H7422-40 is an excellent detector for all TCSPC applications where sensitivity has a higher priority than time resolution. The typical applications are TCSPC laser scanning microscopy, single-molecule spectroscopy, and FCS. 

W. Becker, K. Benndorf, A. Bergmann, C. Biskup, K. Konig, U. Tirlapur, T. Zimmer, FRET measurements by TCSPC laser scanning microscopy, Proc. SPIE 4431, 94-98 (2001)... 

With a dead time of 100 ns per TCSPC channel, total useful count rates of the order of 20 MHz can be achieved. All four channels can be used for multidetector operation. The high count rate and the high number of channels make multimodule TCSPC systems exceptionally useful for diffuse optical tomography [34], and high count rate applications in laser scanning microscopy [39]. Details are described under Sect. 5.5, page 97 and Sect. 5.7, page 129. 

Applications of multiwavelength TCSPC to laser scanning microscopy have been demonstrated in [35, 60]. Spectrally resolved detection in diffuse optical tomography is described in [23]. A multianode MCP PMT and an SPC-330 TCSPC module were used to resolve the luminescence of alkali halides under N, Ar, Kr, and Xe ion irradiation [266]. 

---