Side scatter

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

A flow cytometer equipped with forward-scattering and side-scattering detectors and a sorting option that can distinguish cell size-and-shape, sorting specified cells of 1 to 3pM length into small volumes of culture broth in individual plate wells. (This instrument is used for step 2 and in another mode may contribute to step 1.)... 

The flow cytometer, fitted with both forward and side scatter detectors, generates a 2D plot indicating the distribution of light intensity, forward scatter (FS) versus side scatter (SS), and showing the physical profile of the particle responsible for the scatter. Figure 5.3 is such a plot for a mixture of five rod-shaped bacteria from our laboratory. The different strains appear in partly separated clusters (indicated by square boxes and dot color) along the side-scatter axis in the lower part of the plot. 

For cell isolation purposes an important cytometer sort feature is pulse-pileup also referred to as peak-pileup or PPU. This recognizes split peak intensities arising from two or more cells in the same droplet that are strung together in chains or that coincidentally partially eclipse the laser beam. The two split peaks would each have the same forward- and side-scatter... 

FSC]), strongly related to cell size) against light scattered at a wide angle (side scatter [SSC], which is influenced by cell structure and granularity). 

Sort cells with a Cytomation MoFlo cell sorter with the following parameters forward scatter, side scatter, 730 (LIN mode, amplification factor 6) FL1, 600 (LOG mode) FL2, 600 (LOG mode) trigger parameter, side scatter. The sample flow rate should be adjusted to an event rate of approximately 30 000 s-1. Adjust the sorting gate so that approximately 0.1% of the cells fall within the positive window. 

Log amplifiers are usually employed to analyze fluorescence signals from cells with stained surface markers, because these cells often exhibit a great range of fluorescence intensities. Linear amplifiers are usually employed for analyzing the DNA content of cells, because the DNA content of cells does not normally vary by more than a factor of 2 (e.g., during cell division). Linear amplifiers may be used to analyze forward and side scatter signals, but practice here is apt to vary from lab to lab. With either linear or logarithmic amplification,... 

Fig. 4.3. Different methods of plotting one set of two-dimensional (forward scatter vs. side scatter) data. A Two separate histograms, a dot plot, a three-dimensional plot, and contour plots according to two different plotting algorithms. B Four additional contour plotting algorithms for the same data.

Fig. 5.7. A filter and mirror configuration for making five photodetectors specific for registering forward scatter, side scatter, green, orange, and far-red signals.

Side scatter light (488 nm) will be reflected out of the beam path toward the first PMT by the 500 LP mirror... 

---