22
in the alignment of the excitation light relative to the flow and makes the instrument more
susceptible to mechanical shock and vibration. Reduction of the sample flow means that for
a given concentration of cells, fewer cells can be measured per unit time. Increasing the
sheath flow means that the flow velocity increases, which in turn means reduced sensitivity
(Eq. 1) and increased consumption of water. In any case the width of the focus in the
direction perpendicular to the flow must be considerably larger than the largest cell diameter.
Obviously, one must find a practical compromise between the need for high sensitivity, i.e.
high excitation intensity, on the one hand, and practical values for sample-and sheath flow on
the other.
For instruments designed primarily to measure mammalian cells, which are usually less than
20 /Lm in diameter, the typical compromise is a focal width of about 100 /Lm. A jet in air
(Fig. 8a) usually has about the same diameter and a velocity around 10 m/s. That means a
flow rate of approximately 5 mllmin. For precision measurements, d should not exceed
10 /Lm, which, according to Eq.9, implies that the sample flow, w, must be within 50 /LlImin.
For a sample with 1000 cells/ml this means a measuring rate of 20 cells/min.
Closed flow chambers typically have a cross section of 200 by 300 /Lm. With a sheath flow
of 10 mllmin and d = 10 /Lm this means that the sample flow must be within 16 /L l/min; that
is, significantly less than with a jet in air system. So why not reduce the cross section of the
closed flow chamber? The answer: increased susceptibility to clogging, and larger background
due to scattering from the flow chamber itself.
In order to scatter as little light as possible, the flow chamber should have a minimum number
of surfaces, since scattering arises mainly from impurities and imperfections on surfaces.
Ideally all exposed surfaces should be flat and perpendicular to the direction of the excitation
light. Furthermore, the exposed volume of glass and other materials should be as small as
possible. A jet in air has only two exposed surfaces (the front and the back of the jet), which
are both perfectly clean. However, with its cylindrical shape, the jet in air reflects an
enormous intensity of excitation light over the entire plane perpendicular to the jet, that is,
the plane of the detection optics in laser-based instruments. Hence, the need for obscuration
bar to protect the detection optics. Another factor which may reduce the sensitivity of a jet
in the alignment of the excitation light relative to the flow and makes the instrument more
susceptible to mechanical shock and vibration. Reduction of the sample flow means that for
a given concentration of cells, fewer cells can be measured per unit time. Increasing the
sheath flow means that the flow velocity increases, which in turn means reduced sensitivity
(Eq. 1) and increased consumption of water. In any case the width of the focus in the
direction perpendicular to the flow must be considerably larger than the largest cell diameter.
Obviously, one must find a practical compromise between the need for high sensitivity, i.e.
high excitation intensity, on the one hand, and practical values for sample-and sheath flow on
the other.
For instruments designed primarily to measure mammalian cells, which are usually less than
20 /Lm in diameter, the typical compromise is a focal width of about 100 /Lm. A jet in air
(Fig. 8a) usually has about the same diameter and a velocity around 10 m/s. That means a
flow rate of approximately 5 mllmin. For precision measurements, d should not exceed
10 /Lm, which, according to Eq.9, implies that the sample flow, w, must be within 50 /LlImin.
For a sample with 1000 cells/ml this means a measuring rate of 20 cells/min.
Closed flow chambers typically have a cross section of 200 by 300 /Lm. With a sheath flow
of 10 mllmin and d = 10 /Lm this means that the sample flow must be within 16 /L l/min; that
is, significantly less than with a jet in air system. So why not reduce the cross section of the
closed flow chamber? The answer: increased susceptibility to clogging, and larger background
due to scattering from the flow chamber itself.
In order to scatter as little light as possible, the flow chamber should have a minimum number
of surfaces, since scattering arises mainly from impurities and imperfections on surfaces.
Ideally all exposed surfaces should be flat and perpendicular to the direction of the excitation
light. Furthermore, the exposed volume of glass and other materials should be as small as
possible. A jet in air has only two exposed surfaces (the front and the back of the jet), which
are both perfectly clean. However, with its cylindrical shape, the jet in air reflects an
enormous intensity of excitation light over the entire plane perpendicular to the jet, that is,
the plane of the detection optics in laser-based instruments. Hence, the need for obscuration
bar to protect the detection optics. Another factor which may reduce the sensitivity of a jet