Illumination fluorescence line

We developed two kinds of multidimensional fluorescence spectroscopic systems the time-gated excitation-emission matrix spectroscopic system and the time- and spectrally resolved fluorescence microscopic system. The former acquires the fluorescence intensities as a function of excitation wavelength (Ex), emission wavelength (Em), and delay time (x) after impulsive photoexcitation, while the latter acquires the fluorescence intensities as a function of Em, x, and spatial localization (%-, y-positions). In both methods, efficient acquisition of a whole data set is achieved based on line illumination by the laser beam and detection of the fluorescence image by a 2D image sensor, that is, a charge-coupled device (CCD) camera. 

The fluorescence image of the line illumination on the sample was relayed to the entrance slit of a polychromator. The slit width was set at 70 pm, corresponding to 7 pm on the sample in this configuration. Fluorescence passing through the entrance slit was spectrally dispersed by the grating and detected by a CCD (480 x 640 pixels)... 

Figure 32.2 Experimental set-up for time- and spectrally-resolved fluorescence imaging based on the line illumination.

In this section, based on the methodology presented in the previous section, we describe multidimensional fluorescence imaging and its application to tracking cell responses. We developed the time- and spectrally-resolved fluorescence imaging system based on line illumination, which is capable of rapid acquisition of fluorescence intensities as a function of Em, x, and xy-positions. We applied it to the analysis of an induced plant defense response, that is, the accumulation of antimicrobial compounds or phytoalexins, in oat (Avena sativa). 

In addition, the methodology was applied to fluorescence imaging based on the autofluorescence signals of native molecules in cells or tissues. The imaging system is capable of the rapid acquisition of fluorescence intensities as a function of Em, t, and xy-positions, which is achieved by line illumination of the excitation laser beam. We applied this system to the analysis of a plant defense response, accumulation of phytoalexin in oat leaves, induced by elicitor treatment. In oat leaves treated with an elicitor, we successfully observed weakly fluorescent components, one of which possibly originated from avenanthramide A as a phytoalexin, in addition to the strong fluorescence from chlorophyll molecules. 

Another variation of the FRAP technique scans the laser in one dimension across the cell producing a line-scan profile of fluorescence intensity (14). For a round cell whose surface is evenly labeled with a fluorescently conjugated antibody, a line scan typically produces a profile with two peaks of fluorescence, since the laser beam is illuminating more fluorophores at the edges... 

The fluorescent light from the sample is observed at right angles to the illuminating system. It is passed through colored glass or interference filters or a combination of both. This permits the selection of the lines or group of lines to be observed. Filters of sufficient bandwidth are usually... 

Incident Wavelengths below 1910 A. Hikida et al. (471, 471a) have observed a weak fluorescence of the P(v = 9) bands when NO is illuminated by the 1849 A line. Since the incident photon energy is above the dissociation limit of the [i system, the major path must be dissociation. 

In UV-resonance Raman (UVRR) studies, UV lines such as the fourth harmonic (266 nm) of the Nd YAG laser are used for excitation. Under prolonged illumination by focused UV radiation, quartz and other UV-transparent materials tend to become fluorescent. To avoid the use of window materials and to minimize sample damage by strong UV light, several sampling techniques, such as the fluid jet stream technique (60) and the thin-film technique (61), have been developed. 

Figure 5. Visible laser stability. The laser power fluctuations using a 488 nm (blue) and 568 nm (red) lasers were determined using a 10x objective and a Chroma red slide. The fluorescence was sequentially measured every 30 sec (400 times) for total time duration of 3.33 hrs. The variation of the peak to peak using 488 nm or 568 nm excitation was approximately 25%. The fluctuating power intensity line suggests that the system scanning and detection devices are yielding large power fluctuations that will affect the illumination of the sample. The Acousto Optical Transmission Filter (AOTF) is probably contributing to this 488-568 nm sinusoidal pattern.

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