Optics
Scattered light matches the wavelength of the laser light, which is deflected by the particles it
encounters. Scattering depends on a particle’s physical properties, such as size, shape, surface
topography, and internal complexity.
Excitation by the laser light can cause particles to emit fluorescent light from three sources:
n added fluorochromes or dyes
n naturally occurring fluorescence
n biological structures such as mitochondria and lysosomes (autofluorescence)
Emitted fluorescent light is of lower energy (longer wavelength) than excitation light. Mirrors, optical
filters, and lenses direct the fluorescent light to the detectors.
Forward Scatter
Laser light diffracted by particles in the forward direction (just off the axis of the laser beam) is
collected to give an indication of relative differences in particle size. This forward-scattered light (FSC)
is proportional to particle surface area or size. FSC can be used to distinguish debris from cells or
other target particles; it can also be used to generate a doublet discrimination plot that distinguishes
single particles from multiple particles passing through an interrogation point.
The ZE5 Cell Analyzer can include up to two FSC detectors. Both are highly sensitive PMTs with
adjustable voltage.
The default FSC detector measures 488 nm light from 2–18° relative to the laser beam. It can resolve
cells from debris and measure particles from 0.5–50 μm in diameter. Typical uses include generation
of plots of lysed whole blood suspensions to resolve lymphocytes, monocytes, and granulocytes with
high fidelity.
A second optional detector can be configured for small particle analysis or for measuring forward
scatter generated by a different laser. The small particle option can resolve particles as small as 0.3
µm in diameter.
Each FSC detector can be associated with a mechanical, software-controlled 2.0 neutral density (ND)
filter to alter the range of detection sensitivity. This filter can be enabled or disabled using the Everest
PMT Control panel. See PMT and Laser Controls on page 258.
Side Scatter
Light scattered by particles at an angle of about 90° to the laser beams is collected to indicate relative
differences in particle complexity (for example, granularity, membrane structure, and cytoplasmic
constituents). More complex particles usually reflect and refract more light than less complex particles,
User Guide | 37
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