TCSPC multispectral

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 . 

Multispectral TCSPC FLIM by using a polyehromator at the fibre output of a Zeiss LSM510 NLO was demonstrated in [35]. One-photon excitation with a frequency-doubled Ti Sapphire laser was used. The spectmm was detected by a... 

The book does not include data analysis. Analysis of multispectral TCSPC data, diffuse optical tomography data, or fluorescence correlation data differs considerably from traditional fluorescence decay analysis, and many analysis problems are not entirely solved yet. The author believes that data analysis should be the subject of a different book, and leaves this task to someone who is more familiar with it. 

A stopped-flow setup can relatively easily be equipped with multispectral detection. The fluorescence light is emitted from a small spot in the flow channel. Therefore the flow cell can be placed directly in the input slit plane of a poly-chromator. The spectram is detected by a multianode PMT. The photons detected in the spectral channels are recorded simultaneously by a TCSPC device and a router. However, although the implementation is relatively simple, no spectrally resolved TCSPC-based stopped-flow system has yet been described. 

To make multispectral time-resolved fluorescence measurements into a diagnostic tool, the technique must be combined with imaging. Direct imaging techniques by position-sensitive TCSPC are optically easy. They can be used with wide-field illumination and are easily integrated into endoscopes. However, it is difficult to obtain simultaneous spectral and temporal resolution. Several detectors must be used or several images of different wavelength be projected on one detector. True multispectral resolution cannot be obtained this way. 

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

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