Flow cytometry is used extensively for immunophenotyping of circulating white blood cells. First, blood leukocytes are resolved on the basis of size and granularity. The subpopulations of interest are then gated and analyzed for the presence of specific cell surface antigens using antibodies conjugated to fluorescent probes. This technology can be applied to any cell type to analyze extracellular or intracellular proteins. Cells of interest can also be separated (sorted) from the mixed populations and cultured for further analysis.
Various biochemical and functional properties of cells can be analyzed including hydrogen peroxide production, calcium mobilization, membrane potential and intracellular pH. Cellular processes associated with multi-drug resistance of tumors can be characterized using this technology.
Many pharmacologically active agents alter the growth properties of cells. Detailed analysis of the effects of these agents on cell cycle can be conducted in both fixed and viable cells. Mechanisms of apoptosis can be investigated as well as cell cycle dependent expression of specific proteins. Cellular DNA and RNA content can also be simultaneously measured.
Cytometric analysis is not limited to the study of mammalian cells. This technology can also be applied to lower eukaryotes (i.e. yeast) as well as bacteria. Cell cycle kinetics and various aspects of metabolism can be studied. Cells of interest can be sorted and clonally propagated.
The Cytometry Facility currently has two flow cytometers. These instruments are user-friendly and standard protocols are available for various applications. Following a brief tutorial, investigators/students analyze their samples, independently.
“Cytometry is a process in which physical and/or chemical characteristics of single cells, or by extension, of other biological or nonbiological particles in roughly the same size range, are measured. In flow cytometry, the measurements are made as the cells or particles pass through the measuring apparatus, a flow cytometer, in a fluid stream. A cell sorter, or flow sorter, is a flow cytometer that uses electrical and/or mechanical means to divert and collect cells (or other small particles) with measured characteristics that fall within a user-selected range of values.” — from Practical Flow Cytometry, 4th edition, by Howard M. Shapiro —
Confocal microscopy is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures (a process known as optical sectioning) within an object. This technique is used extensively in the scientific and industrial communities.
Light travels through the sample under a conventional microscope as far into the specimen as it can penetrate, while a confocal microscope only focuses a smaller beam of light at one narrow depth level at a time. Confocal microscopy achieves a controlled and highly limited depth of focus.
The principle of confocal imaging was patented in 1957 by Marvin Minsky and aims to overcome some limitations of traditional wide-field fluorescence microscopes. In a conventional (i.e., wide-field) fluorescence microscope, the entire specimen is flooded evenly in light from a light source. All parts of the specimen in the optical path are excited at the same time and the resulting fluorescence is detected by the microscope’s photodetector or camera including a large unfocused background part. In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal – the name “confocal” stems from this configuration. As only light produced by fluorescence very close to the focal plane can be detected, the image’s optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity – so long exposures are often required. To offset this drop in signal after the pinhole, the light intensity is detected by a sensitive detector, usually a photomultiplier tube (PMT) or avalanche photodiode, transforming the light signal into an electrical one that is recorded by a computer.