Abbott CELL-DYN 3200, Руководство оператора системы

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CELL-DYN ® 3200 System Operator’s Manual
9140181H—October 2001
Principles of Operation
Flow Cytometry
Section 3
Detection with the Optical Bench
The optical bench assembly contains the components that make up the flow
cytometer. It is depicted in Figure 3.1. The main purpose of the optical bench is to
detect the light scattered by the cells as they pass through the flow cell. The
detection process is discussed in this section.
The light source is a vertically polarized 10 mW helium-neon laser with a
wavelength of 632.8 nm. The laser beam passes through a cylindrical lens which
changes the shape from a circle to an ellipse. The beam is then directed through a
125 µm slit which blocks the weaker outer edges. This process yields a uniformly
intense beam approximately 80 µm wide that allows the cell stream to wander
slightly in the flow cell and still be exposed to the same light intensity. An imaging
lens centers the focused laser beam onto the quartz flow cell.
The Sample Transfer Syringe injects different sample dilutions into the sheath
stream in the Optical Flow Cell. The sample is hydrodynamically focused into a
small stream approximately 30 µm in diameter. This focused stream aligns the
diluted cells in single file as they pass through the light beam, which allows them
to be detected one at a time in the sensing region of the detectors.
Since the average diameter of the cells are smaller than the focused laser beam, the
cells do not scatter much laser light. If the remaining unscattered light were
allowed to reach the 0° and 10° (forward) detectors, it would saturate the
electronics. Therefore, an obscuration bar blocks 0° – 1° of the forward unscattered
light beam. The forward angles of scatter are directed to a perforated mirror. The
0° (1° – 3°) light scatter passes through the mirror to the 0° silicon photodiode
detector. The 10° (7° – 10° or narrow angle) light scatter is deflected off the mirror
to the 10° silicon photodiode detector.
The orthogonal scatter is directed through a 700 µm slit which blocks the scatter
from the walls of the flow cell. A beam splitter then separates the orthogonal light
scatter into two portions. One portion of the light is directed to the 90° PMT. The
remaining light is directed through a horizontal polarizer. Only light that has
changed polarization (depolarized) can pass through the polarizer to the 90°D
PMT. (PMTs are used because relatively little light is scattered at this angle.)
The light signals collected by each detector are converted into electrical signals or
pulses. The pulses are digitized based on intensity and sorted into 256 channels for
each angle of light measured.
If a pulse falls above the hardware threshold in the 0° and 10° detectors, the cell
counter counts the pulse and stores it for further evaluation. Pulses that fall below
this threshold are not included in the count.
The information from each detector is collected in list mode. This format stores the
channel information from each of the four dimensions. The data is then used to
determine the WOC differential and RBC, PLT, and NOC counts. Data stored on
floppy disks in the list mode format may be reconstructed into scatterplots at any
time or analyzed by different algorithms as revisions are made.