facs_1.jpgSince the seventeenth century, the renowned work of Anton van Leeuwenhoek in Holland and Robert Hooke in England made the diversity and biological significance of the cellular world commonplace in the international scientific community. The reductionist study of the cellular and molecular mechanisms behind fundamental cellular processes, through microscopy, is central to almost all basic science research in biology today.

Yet science has moved beyond the simple and compound light microscopes of the seventeenth century. Transmission and scanning electron microscopes have afforded the scientific community a new level of insight and depth that was absent before the middle of the past century. These developments are well-known and well-utilized, as every other page in high school biology and chemistry textbooks bears an image from an electron microscope. Thus, this technology is generally understood to be the most accurate method of illustrating the micro-environments that provide the backbone for many scientific processes.

One consequence of the documented nature of this technology is that the prominence of fluorescent microscopy and fluorescent cellular analysis in biological research is obscured. Fluorescent imaging and so- called “flow” cytometry, used by over sixty laboratories at Dartmouth Hitchcock Medical Center (DHMC), is relatively nonexistent in undergraduate research at Dartmouth College.

“I don’t know why more undergraduates don’t use it,” said Dr. Alice Givan, associate professor of physiology and director of the Englert Cell Analysis Laboratory. “DHMC labs dominate it, and they don’t have many undergrads” (1).

The Englert Laboratory, located in the Borwell building at DHMC, is a “core” facility that is shared among researchers at DHMC and the college and makes available two separate technologies: fluorescent imaging and flow cytometry. These technologies are similar in that they provide insight into the structural identity of cellular and other microscopic matter. They differ, however, in that fluorescent imaging literally provides an image while flow analysis quantifies the number of identical or similar substances within a sample (1).

The overarching goal of these two technologies is similar. Both imaging and flow analysis rely on the principle that cells with different structures, when placed under a light source, will scatter the light in different ways. Moreover, they rely on the principle that cells bear unique protein markers that can conjugate with antibodies specific to those markers. These antibodies, in turn, can be fitted with fluorescent stains that fluoresce when exposed to light. In other words, the technologies rely on the fact that cells, when stained with fluorescent markers, will fluoresce in the presence of light. For example, a mixed sample of Type A and B cells can be stained with a cocktail of conjugated antibodies that are specific for surface proteins on each cell type. If the antibody specific for cell A is conjugated with green and the antibody specific for cell B is conjugated with red, these two cells can be distinguished when exposed to light (2).

Fluorescent imaging depends on the physical principles behind the scattering of light. In a typical fluorescent microscope, a laser, usually of argon, shines through a sample at a wavelength of 488 nm. The sample, in turn, fluoresces according to how it was stained. Certain cells, and often even specific cellular structures, can be resolved, which results in a fluorescent image that is reflected into an optical lens or screen for visual analysis. This allows a researcher to create an image that is more realistically three-dimensional than what might be generated with a light or electron microscope (2).

Figure 1 shows an image from a study on tumor phagocytosis by Dr. Paul Wallace, Director of the Flow Cytometry Laboratory at the Roswell Park Cancer Institute. Fluorescent microscopy is used to demonstrate how tumor cells can be treated to promote their engulfment by phagocytes. The choice of markers allows researchers to accurately identify the movement of lymphoma cells into the phagocyte. In this case, the red cell is engulfed by the green cell (1).

facs_2.jpgSimilarly, flow cytometry begins with antibody- mediated fluorescent staining. The goal of this technology is to differentiate among different cell populations. As opposed to providing a visible “photograph” of the relevant structural elements as in fluorescence imaging, flow cytometry detects differences in how the cellular bodies scatter light or how stained bodies fluoresce light. In flow analysis, a sample of stained cells or particles is introduced into a flow cytometer. From there, the cells merge with a rapidly flowing stream, called a sheath, which is pumped into a junction with the cells using an electronic motor. The cells then pass through the beam of a fixed laser. Photoreceptors beyond and to the side of the beam detect the nature of the scattered light and fluorescence, which is translated into a scatterplot of cells or particles based on the various wavelengths of the scatter (2). Figure 2, taken from the study by Dr. Wallace of tumor phagocytosis, shows lymphoma cells that were treated with one stain and phagocytes that were treated with another. It was possible to quantify the extent to which phagocytes devoured the lymphoma cells by identifying those conglomerates of cells that reflect or fluoresce light at a wavelength indicative of having the markers for both lymphoma cells and phagocytes (1).

The technology used in flow cytometry has led to a second application. Since it is capable of differentiating among cells or particles on the basis of cell surface protein markers for the purpose of analysis, it is also able to use that information to sort cells based on the presence of certain cell surface markers. This concept is the basis for machines called fluorescent-activated cell sorters, abbreviated FACS.

It must be noted, however, that both machines that simply analyze cellular structure and those that sort according to this structure are called FACS, mainly because the sorting application was developed prior to the analysis application. The sorting begins with the same steps utilized in analysis. The stream of cells, however, is passed through a vibrating mechanism, which is timed to separate the stream into individual drops with about one cell per drop. These drops, in turn, are assigned electrical charges commensurate with their type. In other words, cells of different types, as determined by their unique light scatter patterns, are given different charges. The drops of cells then fall between two charged deflection plates. As a result of the charges being assigned differently, cells of the same type are deflected in the same direction, while cells of different types are deflected in other directions (2).

This process is illustrated by the following example: a researcher has a sample of lymphocytes but specifically wants to isolate γδ T cells (a specific subset of T cells that have a unique structure and play a prominent role in recognizing lipid antigens). The cell sample can be stained with a fluorescent antibody that can only bind with specific surface markers on γδ T cells. In the scatterplot of the cells arranged by wavelength of scattered light, the researcher can then select those cells that are positive for a specific marker or combination of markers if the cells had been doubly stained. Once “gated,” the sorter can then assign a charge only to the cells of interest. These cells are deflected in one direction once they pass between the charged deflection plates, while the other cells go in the opposite direction. The γδ T cells are then collected in a new sample tube, having been directly isolated from a much more diverse collection of cells.

While this technology is amazingly accurate, it is certainly not flawless. Using round, polystyrene beads instead of cells, the machines may only recover 95% of those selected. Moreover, a 90% recovery rate is often seen due to the fact that irregularly shaped cells may clump together (1). In other experiments, recovery of cells may be very low due to the fact that those cells need a complex set of growth factors to remain viable in vitro for a long period of time. Nonetheless, Dr. Givan asserts that FACS is an invaluable resource. “It is interesting because of the combination of different scientific fields, from chemistry and physics to biology,” she said (1). Thus, the 488 nm wavelength is bridging the scientific gap between the micro and macro worlds.

References:
1. A. Givan, Personal interview, 22 Nov. 2006.
2. A. Givan, Methods in Molecular Biology: Flow Cytometry Protocols, Ed. T S. Hawley and R G. Hawley, 2nd ed (Totowa: Humana P.) 1-10.