Lasercapture microdissection is a technique used to isolate specific populations of cells from an intact tissue sample or histological specimen after visualizing the cells by light microscopy. The cells of interest can be ‘captured’ by placing a polymer over the sample on the microscope slide, and using an infrared laser to melt the polymer onto the sample in discrete locations. Once completed, the polymer–cell composite can be lifted and DNA, RNA, or proteins can be isolated from the dissected cells (Fig. A.20). A variant of this approach makes use of an ultraviolet (UV) laser, rather than infrared. In this case, the UV laser acts as a molecular cutting tool, and can be used to cut away or even ablate the unwanted portions of the tissue, leaving intact the area of interest.
A-18 Flow cytometry and FACS analysis.
An immensely powerful tool for defining and enumerating populations of immune cells is the flow cytometer, which detects and counts individual cells passing in a stream through a laser beam. A flow cytometer equipped to separate the identified cells is called a fluorescenceactivated cell sorter (FACS). These instruments are used to study the properties of cell subsets that are identified by using monoclonal antibodies against cell-surface or intracellular proteins. Individual cells within a mixed population are first labeled by
Fig. A.20 Laser-capture microdissection. Specific populations of cells from an intact tissue sample or histological specimen can be isolated after visualizing the cells by light microscopy. A polymer called the transfer film is placed over the sample on the microscope slide, and an infrared laser is used to melt the polymer onto the sample in discrete locations. The polymer–cell composite is then lifted and the cells of interest are isolated. DNA, RNA, or proteins can be prepared from the dissected cells.
treatment with specific monoclonal antibodies coupled to fluorescent dyes, or by specific antibodies followed by fluorescently tagged anti-immunoglobulin antibodies. The mixture of labeled cells is then suspended in a much larger volume of saline and forced through a small nozzle, creating a fine stream of liquid composed of droplets, each containing a single cell. As each cell passes through a laser beam it scatters the laser light, and any dye molecules bound to the cell are excited and fluoresce. Sensitive photomultiplier tubes detect both the scattered light, which gives information about the size and granularity of the cell, and the fluorescence emissions, which give information about the binding of the labeled monoclonal antibodies and hence about the expression of cell-surface or intracellular proteins by each cell (Fig. A.21).
In the cell sorter, the signals passed back to the computer are used to generate an electric charge, which is passed from the nozzle through the liquid stream at the precise time that the stream breaks up into droplets, each containing no more than a single cell; droplets containing a charge can then be deflected from the main stream of droplets as they pass between plates of opposite charge, so that positively charged droplets are attracted to a negatively charged plate, and vice versa. Once deflected, droplets containing cells are collected in tubes. In this way, specific subpopulations of cells, distinguished by the binding of the labeled antibody, can be purified from a mixed population of cells. Alternatively, to deplete a population of cells, the same fluorochrome can be used to label different antibodies directed at marker proteins expressed by the various undesired cell types. The cell sorter can be used to direct labeled cells to a waste channel, retaining only the unlabeled cells.
When cells are labeled with a single fluorescent antibody, the data from a flow cytometer are usually displayed in the form of a one-dimensional histogram of fluorescence intensity versus cell numbers. If two or more antibodies are used, each coupled to a different fluorescent dye, then the data are usually