ChemoMetec SP-100 - Manual (Page 24)

1 Basics of the SP-100 system
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exclude external light from interfering with the analysis. The measurement is activated,
by pressing the “
Run” button.
When “ Run” is activated, the actuator moves the piston rod down through the barrel of
the cassette. Hereby, the stained lyzate mixture is transported through the flow system
of the cassette. The form and dimensions of the flow channels facilitate effective mixing
of lyzate mixture and stain. Sensors monitor liquid flow, and when the stained lyzate
mixture has reached the measurement chamber of the cassette, the actuator stops the
piston movement.
A standing still situation is obtained after a few seconds. Then the stained mixture of
the measurement chamber is illuminated by green excitation light from eight LED's. The
green light is passed through an excitation filter before it reaches the measurement
chamber. The excitation filter allows only green light of the appropriate wavelengths for
the excitation of PI to pass.
The green light will excite the PI bound to DNA of the sperm cells and in return PI emits
red fluorescence light. Some of the green light will pass through the measurement
chamber together with the red fluorescence light. This green light is now absorbed in an
emission filter, which only allows the red light to pass through. Using an optical lens
system the red light is finally focused on the CCD chip of the camera.
When the red light reaches the CCD, the photons are transformed into electrical signals.
A fluorescent image of the content of the measurement chamber is recorded from the
electrical signals and an example of such an image is shown in
Figure 9 . Each spot in
the image represents a sperm cell nucleus stained with PI. The spots are red but they
are represented as white dots in the image because the camera has a black and white
CCD.
Figure 9. Fluorescent image (from SemenView PC software) of sperm cell nuclei
contained in the measurement chamber of an SP1-Cassette