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Install Cross Polarizing Light Filters if necessary
Under cross-polarized light (CPL), particles that exhibit birefringence are easily visible (as white, grey,
and black shapes on a dark background). These particles are captured by VisualSpreadsheet and the
images are saved as a collage file.
A FlowCAM-XPL model (or optional XPL upgrade) utilizes two cross polarizing filters to properly
detect particles that exhibit birefringence when subjected to cross-polarized light (CPL). One of the
filters is fixed and the other filter is adjustable (via an index). With both filters installed, the second
filter may be rotated and adjusted. As this filter is moved, its orientation will allow a varying degree of
light to pass through the flow cell and on to the first filter and camera. The screen will fade from light to
dark and back again during one complete rotation of the second filter. It can be confirmed that the
darker setting corresponds to the proper value by observing the Pixel Intensity Mean reading in the
Setup & Focus window.
VisualSpreadsheet must be configured to properly work under cross-polarized light conditions. A
FlowCAM-XPL will have appropriately adjusted Factory Default files for use under CPL. To optimize
and detect the birefringence of the particles of interest certain context settings must be employed. The
primary context setting that is adjusted is the Particles Defined By parameter. This parameter is found
via Main Window > Setup > Context > Capture (tab) > Particles Defined By (section). The user should
select the Light Pixels option. This is due to the fact that birefringent particles of interest will appear
white against a dark background. Users can employ the standard tools available in VisualSpreadsheet to
remove any captured images that are clearly not a particle of interest. The development and
implementation of custom filters can also increase the efficiency of this process.
Installing a Flow Cell
Remove the Flow Cell Holder from the Flow Cell Holder Mount (see Figure 2, letter L) by loosening the
thumb screw by turning counter-clockwise. Unscrew and remove the retaining cap from the base of the
Flow Cell Holder. This is accomplished by turning the cap counter-clockwise.
Place the appropriate flow cell into the slots of the Flow Cell Holder. Typical Objective/Flow Cell
combinations are as follows (see Appendix I for more information): 20X/50 µm, 10X/100 µm, 4X/300
µm, and 2X/800 µm. Make sure that the proper flow cell holder is used for each flow cell size.
Re-install the retaining cap onto the Flow Cell Holder by turning the cap clockwise. Gently hold the
flow cell straight when tightening the cap. The cap is sufficiently tightened when the flow cell cannot
be moved . Do not over-tighten the Flow Cell Holder cap, especially when using the 50 µm or 100 µm
flow cell. Excess pressure can break the glass flow cell. Re-install the Flow Cell Holder onto the Flow
Cell Holder Mount and tighten the thumb screw (Figure 6).
TIP:
When using a 100 µm flow cell, place
the flow cell on a surface that allows the flow cell to be fully supported in a straight line. When lifting
the flow cell, think about the pressure that is put on the junction of the tubing and the flow cell. Avoid
putting stress on this area, as it will increase the chances of breaking the flow cell.
