Fluorescence channel

When the coulometric detector was turned on, both leuco forms were completely oxidized to their nonfluorescing chromatic forms and thus vanished from the fluorescence channel. This disappearance was balanced by the arrearance of their chromatic forms in the diode array channel. The confirmation of malachite green, gentian violet, and tlieir leuco analogs in catfish and trout tissue could be based, therefore, on the correct retention times, the observation of the natural fluorescence of the leuco forms when the coulometric detector was turned off, the absence of the leuco form peaks in the 588 nm channel when the coulometric detector was off, the disappearance of the fluorescence of the leuco forms when the coulometric detector was on, the appearance of peaks of parent drugs formed by oxidation of the leuco forms in tlie. S88-nm diode array channel, and the correct ultraviolet-visible spectra maxima for all four peaks. 

Adjust the photomultiplier high-voltage setting to ensure that the bead signal(s) appears in standardized channels. Figure 1 illustrates the distribution of FluoSpheres (Dako) m the green fluorescence channel (FL1, FacScan, Becton Dickinson). 

Run tubes through the flow cytometer, recording FSC, SSC, fluorescence 1 (FITC and fluorescence 2 [PE]). Fluorescence channels should be set to log amplification in this experiment, the flow cytometer was set to conditions routinely used for analysis of lymphocytes. 

Determine mean fluorescence channel number in PE channel... 

Note that any change in the photomultiplier voltages of the fluorescence channels requires the compensation to be readjusted using the above procedure. 

Fig. 4.4.3. Covalent labeling of nuclear targeted wl60hAGT-NLS3 in AGT-deficient CHO cells. Confocal micrographs A-C show overlays of transmission and fluorescence channels (exc 488 nm). The size bar in A-C corresponds to 10 pm. Confocal micrographs (A-C) illustrate the time course of the labeling of transiently expressed wl60hAGT-NLS3 with BGAF in AGT-deficient CHO cells. (A) AGT-deficient CHO...

Cell samples are analyzed on a Becton Dickinson FACScan with a linear fluorescence channel where the fluorescence is proportional to F-actin content. Samples were excited by an argon laser at 488 nm and emission was measured at 525 nm (green fluorescence/FL1) (7). 

For comparisons of genome size, the Gt peaks from the species of interest are compared. Initially, a species should be selected as a standard. The fluorescence channel or fluorescence intensity of nuclei is not a meaningful number unless it is compared relative to a standard. Therefore, each day an analysis is run, at least two samples of the standard should be examined. The flow cytometer parameters are set such that all analysis will... 

Fig. 10.11 Adsorption of 3LNPs with PKH26 on SKOV-3 cell membrane at pH 7.4 (a, b) and 6.0 (c, d) at 4°C observed with confocal scanning laser fluorescence microscopy. Differential interference contrast (a, c) and red fluorescence channel (b, d)...

A laser scanning ophthalmoscope can relatively easily be combined with the TCSPC scanning technique (see Sect. 3.4, page 37). The fluorescence light from the retina is split off by a dichroic mirror and detected by a second PMT. The detection wavelength of the PMTs is selected by filters, FI and F2. The photon pulses from the fluorescence channel PMT are fed into the start input of the TCSPC module. The stop pulses come from the diode laser. 

Each scan is processed to obtain an absorption scan ln(/o//,), ln(/,//2), and F//fl for the fluorescence channels. In biological applications, analyzing the fluorescence excitation spectrum FUq is the method of choice, and the methods of data analysis pertaining to such a method of data collection... 

In both TIRF and spinning-disc microscopy, there are two ways of acquiring multiple fluorescence channels in order to relate the localizations of different proteins to each other the images can be acquired either consecutively or simultaneously. Requirements and shortcomings of these modes of imaging are outlined as follows. 

Keep in mind that the number of fluorescence channels and z-planes collected as well as the number of stage positions determine the duration of each step interval. Ideally, dendritic cells are recorded with one frame per minute. Slower acquisition is possible but might restrict semiautomated cell tracking (see step 2 in Section 8). 

Another important advantage of polychromatic flow cytometry is the measurement of rare cell populations. Multichannel detections allow applying the so-called dump channel. Undesired cells can be excluded from analysis using this dump channel after their staining by specific antibodies, labeled with the same fluorochromes. Using the other fluorescent channels (dyes), low-frequency cell subsets can be detected in a very sensitive manner. 

Fig. 2 Detection of living algae (green line, control), d3dng and dead algae black line, heated sample) [16]. Emission spectra of Chlorella-like. algae ate shown with or without heat treatment. Yellow and pink areas represent each range of detection for the yellow or the red fluorescence channel used for the following flow cytometry...

Fig. 5 Overview of FCM used for this study. Algal cells that had passed through a capillary with 100-pm round bore were analyzed. The FSC signals, the fluorescence of endogenous chlorophyll in the red fluorescence channel and that in the yellow fluorescence channel were collected simultaneously...

Asses scanner performance. The easiest way to test the scanner function is to make up a test slide with Cy dye spots. Make a dilution series of Cy5 and Cy3 spots and apply the Cy dye spots with a spotter. Homemade spotters are easily prepared by drawing a glass capillary. When scanned, these should show that both fluorescent channels are being read. It is also useful to scan slides in both orientations (provided the first scan has not bleached the entire slide). Any spatial variation should be reproducibly at the same position on the slide surface regardless of scan direction that is, ratios should be the same with each scan. 

Figure 7.12 (a) Histograms illustrating the lack of effect of DNIC with glutathione (1 20 DNIC) on the state of DNA in HeLa cells, the lack of the HeLa subpopulation with the DNA content less than the diploid level (<2c, late apoptosis) on the fluorescence channels with the channel numbers <75. DNIC concentration - 100 pM (i), 200 pM (ii), and 500 pM (iii). The cells were incubated for 22 h in Eagle s medium supplemented with fetal calf serum. Solid line Control. Dotted line Incubation with DNIC. -2c- and —4c- Subpopulations of cells with the diploid and tetraploid content of DNA, respectively, (b) Histograms illustrating the pro-apoptotic effect of DNIC with... 

The hybrid K-edge densitometer (HKED) turned out to find quicker applications for safeguards verifications (Ottmar et al. 1986 Ottmar and Eberle 1991) at the input of spent fuel reprocessing plants, as the sample needs neither a dilution nor any treatment before measurement. The technique, considered as an NDA method and described in Sect. 63.3.1, is installed in particular in on-site analytical laboratories and operated by analytical chemists on duty there. Operators have found the instrument so practical and reliable that they use it in their nuclear material accountability program (Brousse et al. 1993). The HKED fluorescence channel is also used for DA of Th or mg-size Pu samples. 

Scan gel on a Typhoon fluorescence scanner using the Cy3/ rhodamine fluorescence channel (see Note 16). 

Fig. 20 Copper bis(thiosemicarbazonato) complex designed by Pascu et al. imaged in HeLa cells using confocal microscopy, where a) is the fluorescent channel, b) the DIC and c) the overlay of each channel (Ref 127).

Figure 11.15 Fluorescent protein as a mechanophore at the fibre-epoxy resin interface in self-reporting fibre-reinforced composites, (a) The formation of microdamages promotes interfacial debonding between resin and fibre, therefore causing the protein to unfold and to lose its fluorescence. (b) Confocal fluorescence microscopy image of a damaged glass fibre-eYFP/epo>y composite, (c) Z-stack projection of confocal fluorescence microscopy images of a damaged carbon fibre-eYFP/ epoxy composite. (F yellow fluorescence channel, O overlay of fluorescence and transmission images).

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