Manual

2 Introducing the NucleoCounter
10
2.1.1 Fluorescence microscope
The compact fluorescence microscope integrated in the NucleoCounter comprises LED´s
(light emitting diodes) as excitation light source, excitation and emission filters, optics
(lenses) and a CCD (charged coupled device) camera. Furthermore, the NucleoCounter
contains advanced software for image processing. The fluorescence microscope, with a
cassette inserted, is shown in Figure 3.
Figure
Figure Figure
Figure 3
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3.
..
. The NucleoCounter´s compact fluorescence microscope
Before the NucleoCounter fluorescence microscope is activated a NucleoCassette is
loaded with sample and inserted into the insertion slit of the instrument. The lid is
closed in order to 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 cylinder
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 the liquid flow, and when
the stained lyzate mixture has reached the measurement chamber of the cassette, the
actuator stops the piston movement.
The measurement chamber is illuminated by green excitation light from light source.
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 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 removed by 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.