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Operation Principles
Cell Illumination
Figure 3.2 Laser Beam Shaping
1. Violet laser beam
2. Blue laser beam
3. Red laser beam
Cell Illumination
As cells in the sample stream go through the sensing area of the flow cell, the elliptical beam
illuminates them. The cells scatter the laser light and emit fluorescent light from autofluorescence
and the fluorescent dyes attached to them.
Forward Scatter
The amount of laser light scattered at narrow angles to the axis of the laser beam is called forward
scatter (FS). The amount of FS is proportional to the size of the cell that scattered the laser light.
Side Scatter and Fluorescent Light
The amount of laser light scattered at about a 90° angle to the axis of the laser beam is called side
scatter (SS). The amount of SS is proportional to the granularity of the cell that scattered the laser
light. For example, SS is used to differentiate between lymphocytes, monocytes, and granulocytes.
In addition to the SS, the cells emit fluorescent light (FL) at all angles to the axis of the laser beam.
The instrument measures the amount of FL emitted by cells depending on the reagents used. For
example, FL above the background FL is used to identify molecules, such as cell surface antigens.
Light Collection, Separation and Measurement
Forward Scatter Collection
The FALS (Forward Angle Light Scatter) detector collects scattered light from a particle that
intersects with a laser and delivers information roughly proportional to the size of the particle. The
3-4
4. First stage shaping lens
5. Second stage shaping lens
6. Flow cell
B49006AS
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