Biomedical Engineering Reference
In-Depth Information
Higher analysis rates are limited by detector sensitivity, data acquisition electronics,
and cell coincidences. The stochastic arrival of particles in the detection volume
limits their concentration to avoid an intolerable number of coincidences.
The cost, complexity, and size of existing flow cytometers preclude their use
in point-of-care (POC) diagnostics, doctor's offices, small clinics, on-site water
monitoring, agriculture/veterinary diagnostics, and rapidly deployable biothreat
detection. The conventional design of flow cytometers is not readily extendable
to applications where high performance, robustness, compactness, low cost, and
ease of use are required in a single instrument. To date, all fluorescence-based
flow cytometers employ the same basic optical configuration, namely, intense
illumination of the bioparticle as it speeds through a highly localized light spot,
generally generated by a laser [ 2 , 4 ], an elaborate arrangement of precision optics,
and sensitive detectors to record fluorescence and scattered light.
The excitation region covers usually the lateral width of the flow channel and
expands some tens of micrometers along the flow direction. A number of commer-
cially available flow cytometers use multiple excitation sources, each focused on a
well-defined location or region separate from the others.
The detection region(s) in flow cytometers are commonly defined by - not
necessarily diffraction limited - confocal light collection optics with high numerical
aperture lenses. Light emitted from each source's region is typically analyzed with
a series of dichroic beam splitters, filters, and photomultiplier tubes (PMTs) in
order to detect and distinguish differently stained particles including those that
simultaneously carry multiple dyes.
The exciting light spot, the detection area, and the particle stream need to reliably
overlap in any flow cytometer. Therefore, the size, position, and flow speed of the
particle stream need to be accurately controlled, which is typically realized by
hydrodynamic focusing. A common implementation of flow focusing is the use
of sheath flow, where buffer liquid surrounds the analyte and thereby effectively
dilutes the sample, lines up the particles, prevents channel clogging, and maintains
clean channel walls. However, the sheath-flow flux can be thousands of times higher
than the analyte flux. Therefore, the necessity for large amounts of sheath liquid and
waste makes the use of sheath flow impractical for POC testing.
In sheath-flow systems, particles travel at a speed of up to several meters per
second resulting in transit times of microseconds. This requires the use of expensive,
high-power, low-noise lasers and high-speed data systems, which increases the cost
and power requirements of a flow cytometer. In addition, since the detection region
is small and the objects traverse it rapidly, such flow cytometers have serious signal-
to-noise ratio (SNR) limitations for weakly fluorescing cells. These limitations
become more acute if multiple targets must be characterized and distinguished for
counting or sorting.
A major cost associated with the use of flow cytometers applied for clinical
diagnostics applications is the cost of reagents (e.g., antibodies and conjugated
dyes). There are two ways to reduce the amount of consumables: first, one can
reduce the required amount of analyte (e.g., by employing microfluidic techniques),
and second, one can reduce the amount of consumable per analyte volume which
requires improved signal-to-noise discrimination.
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