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Frequently Asked Questions (FAQ)
Q.
Do I need to use a collimator or light diffuser with the particular objective I am using?
A.
Review the documentation that came with the instrument to determine the proper configurations.
While the 2X Objective may require the use of a light diffuser, and the 10X and 20X Objectives usually
require a light collimator, it ultimately depends on the specific configuration of your instrument.
Q.
How do I prevent particles that lie over the edge of the camera field of view from getting
imaged?
A.
If your “acceptable region” is unconstrained (for example: Left = 0, Right = 1279, Top = 0,
Bottom = 959), particles that lie across the edge of the field of view will be imaged as a partial particle.
Once you constrain a side by at least one pixel (for example: Left = 1, Right = 1278, Top = 1, and
Bottom = 958), any particle that lies across that boundary will NOT be considered a particle, and
therefore, will not get imaged.
Q.
How can I test the effects of changing the value for the “close holes” function?
A.
The best way to demonstrate close small holes is to do a run of calibration beads, using dark
pixels only and without close holes enabled (i.e., close holes iterations = 0). Be sure to save binary
collage images and raw camera images for this run. Use the binarization overlay to demonstrate that the
software did not use the pixels at the center of the image of a bead (because those pixels are lighter than
the background calibration image). Now go into File Processing Mode and reprocess the raw images
you collected, this time with close holes enabled (i.e., close holes iterations > 0). Repeat this process
using more and more iterations of the close holes algorithm to show how the “hole” is filled (i.e. more
and more of the pixels that are at the center of the bead are being used when calculating particle
properties).
Q.
How is the size calibration determined?
A.
The calibration constants are determined during the manufacturing of the instrument and should
never need to be changed. Fluid Imaging Technologies, Inc. uses a proprietary optical calibration device
to determine the calibration factor of each objective for every instrument. The only time a unit should
be recalibrated is if a major component along the optics path inside the instrument is replaced or
damaged. In that specific instance, the FlowCAM should be returned to Fluid Imaging Technologies.
The size calibration can be verified at any time using a mixture of calibrated microspheres (see
Appendix V). The microsphere and test solutions can be purchased from Fluid Imaging Technologies or
through your sales representative.
