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>Kachel et al. (Cytometry 11:913, 1990) report results from the use of
>a motor-driven chopper disk to interrupt stream illumination to
>generate pulses from a continuous stream of soluble dye in a buffer of
>known pH or [Ca++] to calibrate the cytometer.
>It seems to me that a simpler method would be to use small (e.g. 2
>micron) nonfluorescent plastic beads to trigger the cytometer on
>forward scatter, while using a core/sample stream of diamaeter
>somewhat larger than the particle diameter. ...
The bead trigger sounds like a good idea. It would be
important to distinguish between low level fluoresence
and increased background due to either 90 degree light
scatter from the beads or Raman scatter from the dliuent.
If no information regarding the fluorescent lifetime of the
dye is desired, the type of measurement described above can
be made by using a pulse generator to "trigger" the data
acquisition system. The volume of core stream passing through
the laser per event can be controlled by adjusting the pulse
generator to produce pulses of amplitude and duration
similar to what is observed from a typical cell measurement
event. This measurement can be done on a stained cell
suspension by using light scatter gating to exclude
coincident cell-pulse generator events. It is therefore
possible to gain some insight into how significantly the
free dye fluorescence adds to the overall measurement
background.
When making measurements in this manner, it is important to
understand how the analog signal processing electronics in
the particular instrument used will react to the large DC
component present in this background fluorescence signal. The
chopper method is advantageous in this case; because it
actually produces a pulsed fluorescent emsission.
I've played around with this some and I would be very
interested in hearing about other approaches or ideas.
Gary Durack
Purdue University Cytometry Laboratories
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