Excitation-Wavelength Multiplexing

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]. 

Another, yet more complicated way to record the spectrally split signal is position-sensitive detection by a delay-line-anode PMT [510], or a resistive-anode PMT [262]. 

The lasers must then be multiplexed at a rate faster than the changes expected in the sample. One way to multiplex lasers is to synchronise their pulse periods and delay the pulses of different lasers by different fractions of the pulse period. The fluorescence signals are recorded simultaneously in the same TAC range of a TCSPC device. The principle is shown in Fig. 5.26. 

For a long time the most difficult problem of pulse-group multiplexing has been the lasers. Conventional high-repetition rate lasers cannot be turned off and on at sub-ps speed, and simple optical switches of the required speed and on-off ratio do not exist. Picosecond diode lasers, however, can be switehed on within less than a microsecond and switched off within one or two pulse periods. Diode lasers with multiplexing capability are currently available for 375 nm, 405 nm, 

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Of course, a TCSPC system works effieiently only with a high-repetition-rate excitation source. Diode lasers ean be built with any repetition rate up to about 100 MHz and are available with 375 nm, 405 nm, 440 nm, and 473 nm emission wavelength. Diode lasers are cost-effieient and ean be multiplexed at ps rates see Excitation Wavelength Multiplexing, page 87. For shorter wavelengths, frequency-doubled or frequency-tripled titanium-sapphire or neodymium-YAG lasers can be used. 

Spectral multiplexing or multicolor detection is typically performed at a single excitation wavelength, and relies on the discrimination between different fluorescent labels by their emission wavelength. Desirable optical properties of suitable fluoro-phores are a tunable Stokes shift and very narrow, preferably well-separated emission bands of simple shape. 

Principle used in multidimensional time-correlated single photon counting. Each photon is routed into different memory blocks according to a control signal read synchronously with its detection. Routing is used to record photons detected by several detectors, to multiplex the measurement at different excitation wavelengths or sample positions, or to classify the photons according to an externally measured parameter. 

Multiplexed multidetector systems can also be used in laser scanning microscopy to obtain lifetime images in several emission wavelength intervals and for different excitation wavelength. Please see Sect. 5.7, page 129 and Sect. 5.6, page 121. 

Farrens and Song<40) have replaced the original spark source with a picosecond diode laser in a multiplexed dual wavelength T-formatfluorometer.(41)With an overall instrumental response width of ca. 300 psec full-width half-maximum (FWHM), near-IR fluorescence lifetimes as low as 75 psec in the case of l,l -diethyl-4,4 carbo-cyanine iodide (DCI) (excitation 660 nm) and decay components as low as 48 psec in the case of 124 kDa oat phytochrome (excitation 752 nm) were reported. 

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