Fluorescence histogram

When selecting a particle for analysis, it is important to consider the effects of particle size on fluorescence histogram coefficient of variation (CV). Small particles of uniform size, such as 1 pm diameter latex spheres, exhibit a sharp, narrow histogram with a relatively small CV (Fig. lA). Spherical bacteria. 

Fig. 1. Fluorescence histogram profiles of singlet and doublet 0.92-pm latex particles (A), S. aureus Bioparticles (B), and Zymosan Bioparticles (C).

Fig. 3. Effects of photodetector settings on fluorescence histogram profiles of granulocytes exposed to opsonized 0.92- j,m latex particles. Photodetector settings that are too low (A) or too high (B) are compared with correctly adjusted photodetection (C).

Samples may be evaluated for the percentage of granulocytes with a specific phagocytic load (number of particles per cell) by setting regions for the fluorescence histogram of each population peak. 

The data presented in Fig. 1 illustrate the changes in F-actin polymerization and depolymerization during 5 min of FMLP (UF M) stimulation at 37°C. Human neutrophils often exhibit a biphasic F-actin response under these conditions (18). All of the neutrophils exhibit changes in F-actin polymerization, as illustrated by the fluorescence histograms in Fig. 2, which... 

Fig. 2. Relative F-actin fluorescence histograms of 10,000 neutrophils that are unstimulated (shaded curve) or stimulated with 10 MFMLP for 30 s at 37°C (open curve).

Fig. 2. Fluorescence histogram profiles from the dose-dependent oxidative metabolic responses of grannlocytes stimnlated with 1, 10, or 100 ng/mL of PMA and unstimulated, 2, 7 -dichlorofluorescin-loaded control cells (C). Each histogram represents the analysis of 10,000 cells for each reaction condition.

Fig. 3. Fluorescence profiles of 2, 7 -dichlorofluorescin-loaded cells assayed in whole blood. (A) Compares the fluorescence histograms of unstimulated, control cells (shaded curve) with granulocytes exposed to opsonized S. aureus (open curve). (B) illustrates the two-color analysis profde of the granulocytes that were exposed to Texas Red-labeled S. aureus. Red fluorescence is the result of particle association with each granulocyte, whereas green fluorescence is the result of the oxidation of 2, 7 -dichlorofluorescin to 2, 7 -dichlorofluorescein (DCF). The red and green fluorescence analyses were performed with log-scale detection amplification for each fluorochrome.

To obtain the control and stimulated fluorescence histograms on the same scale, it is usually necessary to analyze the samples with fluorescence log amplification setting on the flow cytometer. 

Fig. 6.4. The fluorescence histogram of an isotype control sample is used to decide on the fluorescence intensity that indicates positive staining.

Fig. 6.6. The fluorescence histogram of a mixture of fluorochrome-conjugated calibration beads and the calibration line for channel numbers and their equivalence in soluble fluorescein molecules derived from that histogram. From Givan (2001).

Fig. 8.1. Propidium iodide fluorescence histograms from nuclei of cells aspirated from normal tissue (upper left) and malignant breast tumors. Data courtesy of Colm Hennessy.

Fig. 8.19. A flow karyotype (fluorescence histogram) of Chinese hamster chromosomes stained with propidium iodide (PI). The G-banded chromosomes from this particular aneuploid cell line are included for comparison with the histogram peaks. From Cram et al. (1988).

Fig. 11.15. A multiplex bead array that can be used to capture multiple soluble analytes. In the dot plot, 15 types of beads are distinguished by their red/orange ratios they capture 15 different cytokines. For example, the beads in Region 1 capture interleukin (IL)-9 in Region 5, IL-2 in Region 9, IL-5 and in Region 13, MCP-1. The green fluorescence histograms from each region show, by their intensity, how much cytokine has been captured by each type of bead. Standard curves can relate the green fluorescence intensity to the concentration of cytokine in the sample. The experimental format illustrated in the cartoon here is patterned directly from work by RT Carson and DAA Vignali (1999).

Since all peripheral blood neutrophils have chemoattractant receptors for N-formyl peptide, the entire population of neutrophils should exhibit a concentration-dependent shift in fluorescence intensity when exposed to FP-Fl. The mean channel values of the normal distribution fluorescence histograms are recorded and plotted vs the concentration of fluorescent FMLPK-Fl (Fig. 2). The data presented in Fig. 2 are from an... 

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