9
FLOW CYTOMETRIC INSTRUMENTATION
Laser-based flow cytometers: The laser-based flow cytometers that are currently commercially
available all have the general optical configuration shown in Fig.I. The laser beam is usually
collimated by means of two lenses, one of which is cylindrical, to form a flat focus of nearly
parallel light. The focus typically has a width of 100 to 200 J.'m and a length in the direction
of the flow of 10 - 20 J.'m. The fluorescence as well as the large angle, i.e. 90", light
scattering are collected by a lens in the direction perpendicular to the flow and the laser beam.
BAND
DICHROIC
FILTERS
LASER
MIRRORS
MIRR~R /
I
~ ~~ / /
ULTRASONIC
/
- PMT
VIBRATOR
0~~~f,"ORESCENCE
~
0
PMT2
DETECTOR (E.G. REO)
SHEATH
SAMPLE
/ -
_
FLUID
/
GtE -FLUORESCENCE
f\
NOZZLE
/ 0
PMT,
DETECTOR (E.G. GREEN)
\.J- - _
~
---RIGHT ANGLE
0V~ ,~" HOLE"
UGH< SCATT"I'G OETEO",
-
LENS
ORIFICE J ET-' - ---.../7\
~
:
\.V-FORWARD LIGHT
" & SCATTERING DETECTOR
-3000V
"
.3000 V
,
, .
• •
~--CELL COLLECTO'S
Figure 1. Laser-based flow cytometer with sorting facilities. Dichroic mirrors separate the large angle light
scattering, which is detected at right angles to the laser beam, and the various spectral components of the
fluorescence. Low angle light scattering is measured by a detector situated close to the forward direction of
the laser beam. Sorting of cells according to the signals they give off as they pass through the excitation focus
is carried out as follows: a piezoelectric crystal on top of the flow chamber oscillates with a frequency which
is typically 40 kHz and thereby causes the jet of water to break into 40 000 droplets per sec just below the
focus. The droplets pass through an electric field of a few thousand V /cm, which is maintained between two
plates. As the droplets are at the point of breaking away from the jet, they are electrically charged either
positively or negatively according to the measuring signal of the cell they contain, by an electrode inside the
nozzle. Thus, they are pulled either right or left in the electrical field according to the fluorescence and/or
light scattering properties of the cell.
FLOW CYTOMETRIC INSTRUMENTATION
Laser-based flow cytometers: The laser-based flow cytometers that are currently commercially
available all have the general optical configuration shown in Fig.I. The laser beam is usually
collimated by means of two lenses, one of which is cylindrical, to form a flat focus of nearly
parallel light. The focus typically has a width of 100 to 200 J.'m and a length in the direction
of the flow of 10 - 20 J.'m. The fluorescence as well as the large angle, i.e. 90", light
scattering are collected by a lens in the direction perpendicular to the flow and the laser beam.
BAND
DICHROIC
FILTERS
LASER
MIRRORS
MIRR~R /
I
~ ~~ / /
ULTRASONIC
/
- PMT
VIBRATOR
0~~~f,"ORESCENCE
~
0
PMT2
DETECTOR (E.G. REO)
SHEATH
SAMPLE
/ -
_
FLUID
/
GtE -FLUORESCENCE
f\
NOZZLE
/ 0
PMT,
DETECTOR (E.G. GREEN)
\.J- - _
~
---RIGHT ANGLE
0V~ ,~" HOLE"
UGH< SCATT"I'G OETEO",
-
LENS
ORIFICE J ET-' - ---.../7\
~
:
\.V-FORWARD LIGHT
" & SCATTERING DETECTOR
-3000V
"
.3000 V
,
, .
• •
~--CELL COLLECTO'S
Figure 1. Laser-based flow cytometer with sorting facilities. Dichroic mirrors separate the large angle light
scattering, which is detected at right angles to the laser beam, and the various spectral components of the
fluorescence. Low angle light scattering is measured by a detector situated close to the forward direction of
the laser beam. Sorting of cells according to the signals they give off as they pass through the excitation focus
is carried out as follows: a piezoelectric crystal on top of the flow chamber oscillates with a frequency which
is typically 40 kHz and thereby causes the jet of water to break into 40 000 droplets per sec just below the
focus. The droplets pass through an electric field of a few thousand V /cm, which is maintained between two
plates. As the droplets are at the point of breaking away from the jet, they are electrically charged either
positively or negatively according to the measuring signal of the cell they contain, by an electrode inside the
nozzle. Thus, they are pulled either right or left in the electrical field according to the fluorescence and/or
light scattering properties of the cell.