20
sensitivity large angle scattering should be measured at relatively low scattering angles, say
around 20°, rather than around 90° which is standard in current laser-based instruments. This
is one reason why an arc lamp-based instrument, which detects large angle scattering upward
from about 15° (Argus 100) exhibits a higher light scattering sensitivity than any of the laserbased instruments that are commercially available.
Flow chambers: The flow chamber is a critical part of the flow cytometer. It is an important
factor in the overall performance of these instruments, and probably the most common source
of trouble in daily routine. There are several types of flow chambers (see Figs. 8 and 9).
Common to all is that they have what is called hydrodynamic focusing, which means that the
sample is introduced into a much larger flow of water, the so called "sheath flow", at a point
where the cross section of the flow is much larger than what it is when it passes through the
excitation focus. Since the flow is laminar and there is essentially no mixing between the
sample and the sheath fluid, the cross section of the sample flow relative to that of the total
flow is maintained as the flow converges toward the excitation focus. Hence, the sample is
confined to a narrow core of the flow. This is done to allow a smaller focus of excitation light
and a correspondingly high excitation intensity. As evident from Eq. 9, there is a
proportionality between the diameter of the sample core, d, and the diameter of the flow, D.
Since the rate of sample flow, w, is always much smaller than that of the total flow, W,
typically by a factor of 100 or more, W is approximately equal to the sheath flow rate
d = (w/W)ll2-D
(9)
In order to achieve a sufficiently high excitation intensity to measure very small and/or weakly
fluorescent cells with adequate sensitivity, the width of the excitation focus should be as small
as possible. On the other hand, since the intensity profile of such a focus is approximately
gaussian, all cells must pass through the central part of the focus in order to be exposed to
the same intensity and therefore to be measured reproducibly. For example, for all cells to
be exposed to the same excitation intensity to within 2 %, they must pass through the center
of the focus to within 10 % of its width. (Focal width is defined as the distance between
points having Ve 2 of the peak intensity). If one allows a 10 % variation in excitation intensity,
the diameter of the sample flow, d, can be increased to 23 % of the focal width.
sensitivity large angle scattering should be measured at relatively low scattering angles, say
around 20°, rather than around 90° which is standard in current laser-based instruments. This
is one reason why an arc lamp-based instrument, which detects large angle scattering upward
from about 15° (Argus 100) exhibits a higher light scattering sensitivity than any of the laserbased instruments that are commercially available.
Flow chambers: The flow chamber is a critical part of the flow cytometer. It is an important
factor in the overall performance of these instruments, and probably the most common source
of trouble in daily routine. There are several types of flow chambers (see Figs. 8 and 9).
Common to all is that they have what is called hydrodynamic focusing, which means that the
sample is introduced into a much larger flow of water, the so called "sheath flow", at a point
where the cross section of the flow is much larger than what it is when it passes through the
excitation focus. Since the flow is laminar and there is essentially no mixing between the
sample and the sheath fluid, the cross section of the sample flow relative to that of the total
flow is maintained as the flow converges toward the excitation focus. Hence, the sample is
confined to a narrow core of the flow. This is done to allow a smaller focus of excitation light
and a correspondingly high excitation intensity. As evident from Eq. 9, there is a
proportionality between the diameter of the sample core, d, and the diameter of the flow, D.
Since the rate of sample flow, w, is always much smaller than that of the total flow, W,
typically by a factor of 100 or more, W is approximately equal to the sheath flow rate
d = (w/W)ll2-D
(9)
In order to achieve a sufficiently high excitation intensity to measure very small and/or weakly
fluorescent cells with adequate sensitivity, the width of the excitation focus should be as small
as possible. On the other hand, since the intensity profile of such a focus is approximately
gaussian, all cells must pass through the central part of the focus in order to be exposed to
the same intensity and therefore to be measured reproducibly. For example, for all cells to
be exposed to the same excitation intensity to within 2 %, they must pass through the center
of the focus to within 10 % of its width. (Focal width is defined as the distance between
points having Ve 2 of the peak intensity). If one allows a 10 % variation in excitation intensity,
the diameter of the sample flow, d, can be increased to 23 % of the focal width.