Biology Reference
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450 nm
470 nm
580 nm
600 nm
Scanning mirrors
Spectral separation
450
500
Wavelength (nm)
550
600
Multianode PMT
Figure 5.12 Simplified scheme of a confocal setup allowing spectral imaging in simul-
taneous mode. Briefly, a laser beam scans the specimen using scanning mirrors. The
pinhole conjugated to the focal plane rejects light emitted by objects outside this focal
plane, which results in optical sectioning. Spectral separation is then achieved by a grat-
ing combined to recycling systems, allowing minimal loss of light. The light is then col-
lected using a multianode PMT. The fluorescence spectrum is recorded for each pixel.
the following sections, the twomost representative techniques, time-correlated
single-photon counting (TCSPC) for TD FLIM and phase and modulation
measurements for FD FLIM are discussed in detail.
4.1.4 Time domain: TCSPC
Most TCSPC systems are implemented on a confocal microscope
equipped with
a pulsed laser source. The source must produce short laser pulses (from
several hundred femtoseconds to picoseconds' width) with a frequency
usually ranging from 10 to 80 MHz. It is interesting to note that the Ti:Sa
laser matches these specifications, which can be of great help in deep and
noninvasive biosensor imaging;
detectors with a fast instrumental response. Optimal instrumental
response function can be obtained using a multichannel plate or the latest
generation of avalanche photodiodes
50 ps full-width at half-
maximum), but they are extremely fragile and require careful handling.
TCSPC manufacturers therefore provide more robust detectors, that is,
optimized photomultiplier tubes, with an IRF of around 250 ps adapted
(
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