Excitation light source

With fluorescence, however, the sensitivity is limited in principle only by the maximum intensity of the exciting light source so that under ideal conditions,... 

Pulsed method. Using a pulsed or modulated excitation light source instead of constant illumination allows investigation of the time dependence of emission polarization. In the case of pulsed excitation, the measured quantity is the time decay of fluorescent emission polarized parallel and perpendicular to the excitation plane of polarization. Emitted light polarized parallel to the excitation plane decays faster than the excited state lifetime because the molecule is rotating its emission dipole away from the polarization plane of measurement. Emitted light polarized perpendicular to the excitation plane decays more slowly because the emission dipole moment is rotating towards the plane of measurement. 

From these vievqjoints, we have developed a femtosecond NIR laser microscope with a home-built cavity dumped chromium forsterite (Cr F) laser as an excitation light source whose output wavelength is centered at 1260 run. In the following the set-up of the NIR laser microscope and its application to multiphoton imaging are presented. 

With the aim of elucidating molecular dynamics in a small domain, we have constmcted several microspectroscopic systems, that is, (i) the confocal microscope with the excitation light source being a femtosecond NIR laser emitting a 35 fs pulse, and (ii) the fluorescence correlation spectroscopic system with optical tweezers. 

Moser, C., Mayr, T. and Klimant, I. (2006). Filter cubes with built-in ultrabright light-emitting diodes as exchangeable excitation light sources in fluorescence microscopy. J. Microsc. 222, 135—40. 

In the CL detection method, the excitation of a molecule is achieved via a chemical reaction that is generally an oxidation process. That is, an exciting light source is not required thus, the CL is not accompanied by any scattering light and source instability. This permits a large signal-to-noise ratio (S/N), which finally provides an increase in sensitivity. 

Instruments. A schematic diagram of the aicrocoaputer-controlled system for the fluorescence measurement just upon ablation is shown in Figure 1. The excitation light source was a 351 nm laser (Lumonics He-400, 15 ns) and fresh surface of PVCz film was examined in air. 

Figure 11.10. Phase-locked detection of fluorescence lifetime using single reference signal. FID = fluorescence inducing and detecting devices LPF = low-pass electronic filter VCO = voltage-controlled oscillator v, = signal to modulate the output intensity of the excitation light source v/= the fluorescence signal.

The computation of far-field radiation from a collection of incoherently radiating dipoles is in general quite a complicated problem. To calculate the angular dependence of the far-field intensity, the volume distribution of excited states must first be obtained, which, as we have seen, depends on the volume distribution of the absorbers and the electromagnetic field which stimulates them. The fields in turn depend on the frequency and linewidth of the exciting light source. Then the emission problem for the excited-state distribution (both spatial and frequency) must be solved including reorientation and depolarization effects. 

If either the protein or ligand is light sensitive then some photolysis ( photodecomposition ) may occur upon exposure to light. Under these circumstances, it is essential to shut off the excitation light source between measurements (usually by closing the excitation shutter that is provided in most instruments). The slit-width on the excitation side can also be reduced in order to minimize overall exposure (this can often be compensated by an increase in the slit-width on the emission side to increase the magnitude of emitted fluorescence). 

Fig. 1. Comparisons of the wide-field, flying spot, pinhole detector, and pinhole confocal microscopes. Components include an excitation light source (V), an excitation filter (E), a dichromatic mirror (DM), an emission barrier filter (B), an objective lens (n), a detector (D), and a pinhole (P).

Another simple modification of the Becquerel phosphoroscope was described by Lewis and Kasha (60). Here one has a cylinder which can be rotated at various speeds with the sample inside. The cylinder contains a window and the exciting light source is placed on one side of the cylinder, with the detector on the other. When the window faces the source the sample is excited, and when it faces the detector the intensity is measured. The time between excitation and detection can be changed by altering the speed of rotation. In this manner the complete decay curve can be plotted. Again it... 

Spectrofluorimeter. A fluorimeter with two separate monochromators. One serves to scan through the spectrum of the exciting light source the other scans the emitted fluorescent light. 

The second type of experimental procedure to observe the spectra of transients uses flash photolysis as illustrated by the work of Porter and Windsor (122). The exciting light source consists of a flash resulting from the rapid discharge of a spark through a circuit having a large capacitance. This first flash pumps the molecules into the excited triplet... 

Work conducted by Tiller and Jones (1997) demonstrated that the fluorescence of PAHs decayed over time under both under anoxic and oxic conditions. Typically, however, the presence of dissolved oxygen had a more pronounced influence on baseline fluorescence decay for all the PAHs studied. Moreover, certain PAHs (pyrene and anthracene) were more susceptible to this phenomenon than others. To date a mechanism to explain this phenomenon has not been identified, but it is probably a combination of complex pathways including the reaction of the analyte with reactive oxygen species formed from the excited triplet state DOM and the direct photolysis of the analyte by the excitation light source. Thus, the application of fluorescence quenching for measuring Kdom is probably limited to systems, which can be analyzed under anoxic conditions. 

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