Time-resolved fluorescence light sources

Finally, the use of CCD cameras as detectors in combination with short-pulsed light sources enables not only spatially resolved imaging, but also a time-resolved fluorescence detection. Time resolution offers two different new alternatives ... 

The light source for time-resolved fluorescence can be a nanosecond pulse of Blum-line nitrogen laser, triple harmonics of yttrium-aluminum-garnet (YAG) laser, second harmonics of mode-locked dye, or gas laser. 

Diuing the past decade, the instrumentation for time-resolved fluorescence of proteins has advanced dramatically. The flashlamp light sources have been replaced by hi -repetition-rate (MHz) picosecond dye lasers, which provide both higher excitation intensities and raon rapid data acquisition. The dynode-chain PMTs have been replaced by MCP detectors, which provide much shorter single-photoelectron pulse widths than a dynode chain PMT. In con nnation. the new light sources and detectors provide instniment r ponse functions with half-widths near 100 ps, so that picosecond resolution can now be obtained. 

A synchronously pumped DCM dye laser was used as the picosecond light source in the pump-probe experiments. A Soleil-Babinet compensator in the excitation beam was set so that the polarization of the excitation and probe beams were parallel or perpendicular. Fluorescence anisotropies were measured with a Spex fluorolog spectrometer. The time resolved fluorescence was measured by single photon counting. 

Although the relation between fluorescence depolarisation and rotational Brownian motion was first identified by Perrin and the development of the theoretical background of the time-resolved fluorescence depolarization experiments was made by Jablonski use of the technique was limited until the advent of improved fluorescence decay time measurements some fifteen years i. An alternative, related technique, involving excitation using a continuous polarised light source, provides only the time average of the correlation function (Eq. 18) and as such, is less useful than the time resolved method. Other disadvantages are that the natural decay time of the chromophore must be determined from a sqrarate experiment and it is necessary to alter the viscosity, and/or temperature of the medium, often withun-... 

The instrumentation for time-resolved fluorescence spectroscopy shares some similarities with that used for steady-state measurements, in the sense that an excitation source and a detection systems are used. The nature of these components may, and usually does, vary significantly. Namely, a pulsed light source is used in time-resolved spectroscopy, which is at variance with the continuous light source used in steady-state fluorescence. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Lasers. Laser sources (discussed earlier in the Spectrophotometry section) are widely used in fluorescence applications in which highly intense, well-focused, and essentially monochromatic light is required. Examples of these applications include time-resolved fluorometry, flow cytometry, pulsed laser confocal microscopy, laser-induced fluorometry, and light-scattering measurements for particle size and shape. Several different types of lasers are available as an excitation source for fluorescence measurements (see Table 3-3). 

Fluorescence can be resolved overtime. The use of very short pulsed light sources (picosecond lasers and laser diodes) has rendered accessible graphs of fluorescence decay as a function of the time. New applications based upon a greater knowledge of the lifetime are under development, although they are still not used much in chemical analysis. 

---