Optical Response

As described in Section 11.2.2, the optical response generated by interaction of a cell with the laser beam at the illumination point consists of forward-scattered light and side-scattered light that are of the same wavelength as the exiting beam. In addition, absorption either by the cellular components or by fluorochromes staining a cell produces fluorescence signals shifted in wavelength from that of the exciting beam. These detected parameters provide information about the type of the cell as well as its structure and function. They are illustrated in Figure 11.6.

The forward-scattered signal (FSC) that is generated within a few degrees from the incident laser beam is often related to the cell size and is used to determine the cross-sectional area and volume of the cell. However, a cell with a very different refractive index from its surrounding can also produce a FSC signal. For example, a dead cell appears smaller than the corresponding living cell because its refractive index is more like the surrounding stream due to the leaky outer membrane. The dead cells therefore bend less light into the FSC detector than the corresponding living cells.

The high-angle scattered signal are often collected at 90°C as a side-scattered count (SSC). This has also been discussed in the previous subsection. This signal is produced by reflection and refraction from the variation in cell structure. Therefore, SSC is related to the cell's surface texture and internal structure. SSC is sometimes also referred to as a granularity signal because it provides information on the granularity of the cell. For example, granulocytic blood cells that have granular and irregular nuclei produce a significantly more intense SSC signal compared to that from more regular lymphocytes or erythrocytes.

Fluorescence labeling allows one to selectively label (stain) a specific subpopulation of cells in order to investigate their cell structures and functions. For example, immunofluorescence, used for immunophenotyping discussed later in Section 11.5.1, involves staining of cells with antibodies which are conjugated to fluorochromes. This staining can be used to label antigens on the cell surface. Alternatively, antibodies can also be directed at targets in cytoplasm. The two approaches used for immunofluorescence are: (i) direct immunofluorescence in which an antibody is directly conjugated to a fluo-rochrome, thus the cells are stained in a single step; and (ii) indirect immuno-fluorescence in which the primary antibody to cell surface antigen is not labeled. Instead a second antibody specific to the primary antibody is conjugated to a fluorochrome.

Modern flow cytometry utilizes a large array of monoclonal antibodies which are specific for various antigen proteins on the cell surface (Givan, 2001). The antigens defined by these antibodies are characterized by a CD number designation where CD stands for "cluster of differentiation," which defines a particular protein differentiating one type of cells from another. The CD numbers now range from CD1 to over 200, as the number of characterized antigens have steadily grown. These CD number designations are used to specify an antibody that is specific to the antigen.

In the direct staining of cells, they are incubated with a monoclonal antibody that has been conjugated to a fluorochrome. This procedure takes only 15 to 30 minutes of cell incubation with antibody at 4°C, followed by several washes to remove antibodies that are weakly bound or bound nonspecifically. In the indirect staining method, the cells are incubated with a nonfluorescent monoclonal primary antibody. Then after washing to remove any weakly bound antibody, a second incubation with an antibody conjugated to a fluo-rochrome is used to react with the primary antibody of the first layer. The advantage of this indirect staining process is the cheaper cost of unconjugated primary antibodies. Another advantage offered by this sequential layer approach is that it can be extended to more than two layers with each layer producing amplification of fluorescence. The disadvantage of this indirect staining process is that it is considerably time-consuming and that complications due to nonspecific binding is significantly increased with each added layer.

Another optical parameter monitored can be the polarization of the scattered light, which provides information on the birefringence produced by the cell structure such as that of eosinophil granules. Furthermore, pulse shape analysis of the scattered signal can be used to get information on the cell shape.

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