Multispectral Fluorescence Lifetime Experiments

Biomedical applications of time-resolved fluorescence often preclude the scanning of a spectrum by a monochromator. The total excitation dose may be limited by photobleaching, or the investigated systems may show dynamic changes in their fluorescence behaviour. For applications in human patients, the laser power is 

Multiwavelength operation of a TCSPC device can be achieved by using a system of dichroic mirrors and a corresponding number of individual detectors (see Sect. 3.1, page 29). The general optical setup is shown in Fig. 5.23. 

A more detailed fluorescence spectrum is obtained by using a polychromator (or spectrograph) and recording the spectrum with a multianode PMT. The principle of a multiwavelength fluorescence experiment is shown in Fig. 5.24. 

The cracial parts of the system are the polychromator and the transfer optics. Polychromators and monochromators are usually optimised for high spectral resolution. This requires keeping the optical aberrations on the path through the polychromator smaller than the slit width. The result is a relatively low f-number, typically 1 3.5 to 1 8. The f-number limits the fraction of the fluorescence light that can be transferred into the entrance slit (see Sect. 7.2.4, page 279). Moreover, the efficiency of any grating is far less than 100%. Therefore some loss of photons on the way from the sample to the detector in unavoidable. A multiwavelength system based on a polychromator is less efficient than a system based on dichroic beamsplitters, but by far more efficient than a system that scans the spectrum by a monochromator. 

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