Flow cytometry scatter

Cledat, D., Battu, S., Mokrini, R., and Cardo, P. (2004). Rice starch granule characterization by flow cytometry scattering techniques hyphenated with sedimentation field-flow fractionation. J. Chromatogr. A 1049(1-2), 131-138. 

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.

Flame Photometry and Gas Chromatography (CyTerra) -Aerodynamic Particle Size and Shape Analysis (BIRAL) -Flow Cytometry (Luminex, LLNL) -Semiconductor-Based Ultraviolet Light (DARPA) -Polymer Fluorochrome (Echo Technology) -Laser-Induced Breakdown Spectroscopy -Raman Scattering -Infrared Absorption -Terahertz Spectroscopy -UV LIDAR... 

Most recent scientific applications involve the determination of direct relationships between input parameters and a known target response. For example, Santana and co-workers have used ANNs to relate the structure of a hydrocarbon to its cetane number,4 while Berdnik s group used a theoretical model of light scattering to train a network that was then tested on flow cytometry data.5... 

Abstract Flow cytometry is a technique for rapidly examining multiple characteristics of individual cells, by recording fluorescence signals emitted from cell-associated reporter molecules, and measuring cellular light scattering properties. This chapter introduces the principles and practice of flow cytometry, and reviews examples from the literature that highlight applications of this experimental tool in the neurosciences. The chapter concludes with protocols for three basic procedures that illustrate some practical aspects of analytical flow cytometry. 

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

Different methods can be used to detect cell death in CLL cells. Methods based on detection of cell death by flow cytometry are recommended as CLL cells are easily distinguishable by forward and size scatter from possible contamination of... 

Four methods of quantitating apoptosis by flow cytometry are described in this chapter. The two-dimensional light scatter assay measures the reduction in cellular volume and increase in cell density that are observed during apoptosis and that are revealed by a decrease in forward light scatter and an increase in... 

The only difficulty in using flow cytometry to monitor cell death is that, as mentioned in Chapter 3, dead cells have different scatter properties than living cells. In particular, because of their perforated outer membrane, they have a lower refractive index than living cells and therefore have forward scatter signals of lower intensity. For this reason, it is important not to use a gate or forward scatter threshold when analyzing a population for the proportion of dead and live cells. Any forward versus side scatter gate drawn around normal lymphocytes, for example, will always show most if not all of the cells... 

Despite these problems, flow cytometry has had some noted success in aquatic research, particularly in relation to studies on the phytoplankton. Because all phytoplankton possess chlorophyll, but only the cyanobacteria possess the phycobiliproteins, autofluorescence signatures from water samples, based on the chlorophyll (fluorescence >630 nm), phycoerythrin (fluorescence <590 nm), and forward scatter of particles, have been used to characterize the changes that occur in plankton at different depths or at different locations (Figs. 11.5 and 11.6). 

Fig. 11.12. The use of gel microdroplets and flow cytometry to assay drug sensitivity of bacterial cells. The figure shows side scatter and green fluorescence contour plots of gel microdroplets (GMDs) containing E. coli cells that have been stained with fluorescein isothiocyanate for total protein. The microdroplets have been analyzed in the flow cytometer either at time 0 or 2 h after incubation in control medium (left plots) or medium containing penicillin (right plots). A model system was created by mixing two strains of bacteria (susceptible or resistant to penicillin). The data show that a small subpopulation of resistant cells could be detected within 2 h because of its rapid growth in comparison to susceptible cells. From Weaver et al. (1991).

Coincidence Coincidence, in flow cytometry, is the appearance of two cells or particles in the laser beam at the same time. The flow cytometer will register these two cells as a single event (with approximately twice the fluorescence intensity and light scatter as a single cell). To avoid coincidence, the concentration of cells in the sample should be low, the laser beam should be small in the direction of flow, and the sample core should be narrow. 

Fixation Fixation is the process by which the protein of cells is denatured, or cross-linked, and preserved. Fixation in flow cytometry is used to inactivate hazardous biological material and also to preserve stained cells when there is not immediate access to a flow cytometer. Fixation is also important in preserving proteins before detergent permeabilization for intracellular staining. Formaldehyde is often the fixative of choice for flow cytometry because it preserves the forward and side scatter characteristics of cells (but does cause some increase in their autofluorescence). 

Lens A lens is a means of changing the shape of a beam of light. In flow cytometry, lenses are used to narrow the laser beam to a small profile at the stream. Some lenses produce a beam with a circular cross-sectional shape others produce beams with an elliptical configuration. Lenses are also used in a flow cytometer to collect scattered light and fluorescence and then to transmit them to an appropriate photodetector. 

Threshold The threshold is an electronic device by which an ADC can be made to ignore signals below a certain intensity. A forward scatter threshold is most commonly used in flow cytometry to exclude very small particles, debris, and electronic or optical noise from acquisition into a data file. 

LASER-BASED FLOW CYTOMETRY AND FORWARD-ANGLE LIGHT SCATTERING... 

Histogram luminescence. Volume 2(11). Single-parameter plot of data. In flow cytometry, the horizontal axis displays the light scatter or fluorescence intensity parameter and the vertical parameter displays the number of events (e.g., cell count). Volume 1(5). 

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