CELL-DYN
®
3200 System Operator’s Manual
3-9
9140181H—October 2001
Section 3
Principles of Operation
Figure 3.2 Optical Flow Cell
Optical Flow Cell
In a flow cytometer, the cell suspension is transferred from the mixing chamber
through a sample tube into a special
flow chamber
with a small opening at the tip. The
suspension is then injected into a stream of fast-moving, cell-free liquid
(sheath
fluid)
. Since the two liquids travel at different rates of speed, they do not
intermingle. The special geometry of the flow cell and the flow rate of the sheath
fluid forces the cells into single file. This process is known as
hydrodynamic
focusing
. (Refer to Figure 3.2 for a drawing of the Optical Flow Cell.)
As the cells enter the
view volume
(specific viewing area), they intersect with the
laser beam. The different types of cells scatter the laser light at different angles,
yielding information about cell size, internal structure, granularity and surface
morphology. The optical signals the cells generate are detected and converted to
electrical impulses which are then stored and analyzed by the computer.
Flow cytometers generally measure two angles of scatter.
Forward angle light
scatter
is a measure of cell size.
Side angle (orthogonal) light scatter
is a measure
of cell surface and internal structure but is primarily a measurement of internal
granularity. Combining the information from the two scatter measurements
provides more accurate discrimination between cell populations than either single
measurement. (See Fig. 3.3 for an example of the light scatter measured by the
CELL-DYN 3200.)
1 Sample Feed Nozzle
2 Sheath Stream
3 Sample Stream
4 Focused Laser Beam
5 Various Angles of
Scattered Light
4
1
5
3
2