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What is Flow
Cytometry? |
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Flow Cytometry refers
to a specific technique wherein the physical and
chemical characteristics of large numbers of cells are
rapidly examined one cell at a time. The basics of flow
cytometry are illustrated below. |
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(1)
Sample Extraction And
Preparation |
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Flow
cytometry can analyze any cell type provided that the cells
are made to be freely suspended in a particle-free saline
solution. Several different techniques are applicable
depending upon the sample type. |
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(2)
Labeling |
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Flow cytometry ultimately
depends upon the presence of specific chemicals in or on the
surface of cells. Cells are small and do not naturally
have enough molecules with strong fluorescence. For this
reason, the cells are specifically labeled with dyes which are
efficient emitters of fluorescence. In order to obtain
specific information about cells the dyes are often attached
to biological molecules which have specific affinity to a
cell’s molecules located on the surface or even within the
cells. The range of labeling techniques currently
available is staggering and is growing daily. |
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(3)
“Single File” Flow |
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When dilute solutions of
these labeled cells are forced to flow through a narrow
stream, the cells are forced to pass in “single file” fashion.
This is typically done by using narrow capillary tubes or
(more conventionally) through the use of hydrodynamic focusing
– surrounding the sample stream with another saline liquid
(cell free) at higher pressure which controllably constricts
the sample stream. |
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(4)
Laser Illumination |
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A laser(s) is focused onto the sample stream in an effort to
illuminate one cell at a time. The dye labels attached to
cells are optically excited and fluorescence is emitted. A
molecule typically emits a number of times depending upon the
speed of the cell and the size of the focused spot. |
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(5)
Fluorescence Collection
And Detection |
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The fluorescence light
generated is emitted in all directions. One attempts to
collect as much of it as possible, filter out the scattered
laser light and detect the rather weak signal using a fast and
sensitive detector (typically a photomultiplier tube). |
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(6)
Data Analyses |
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Fast
detectors and signal processing equipment are critical to flow
cytometry; as many as 100,000 cells/minute are typically
analyzed. Since not all cells of a particular type will have
an identical signal, it is important to employ well-designed
equipment, statistics, and visualization techniques
to make sense of the data.
In practice, a number of different parameters are investigated
simultaneously including the use of more than one fluorescent
label. These multiparameter instruments collect a staggering
amount of data and can be used to learn a great deal about the
sample. |
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Further Reading: |
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As you might suspect, the
design and practical use of these instruments is much more
complicated than described above. For those interested in
learning more about Flow Cytometry, an excellent place to
begin is an introductory text written by Howard Shapiro (Practical
Flow Cytometry,
2nd edition, 1995, Wiley-Liss;
ISBN: 0471303763). |
