Open Cytometry: verifying LED/camera timing with hardware measurements

The open-source cytometer team has been acquiring image series to test image/LED synchronization. The team has verified with a dual-channel oscilloscope and the Acquisition Active pin of the Dart camera that the LED is on during and slightly before and after the camera exposure.

For the next series of experiments:

  1. the UV and blue LED wavelengths illuminate the sample, but these short wavelengths are blocked from reaching the Dart camera by the yellow long-pass interference filter that’s between the camera lenses.
  2. the dye on the beads fluoresces at yellow wavelengths that pass through the yellow filter for measurement by the Dart camera (with dyed malaria DNA, the fluorescent wavelengths will be slightly different but will also pass through the yellow filter)
  3. the measured coefficient of variation (standard deviation / mean) for this cytometer is increased by several factors including: camera noise, camera intensity linearity in space and time, dye variability (one bead vs. another bead, and vs. time), illumination variation in space and time, etc.

To facilitate #3, robust mechanical mounting is a key prerequisite, while allowing for the possibility to reconfigure for iterations undoubtedly necessary.

We start out with one or two sizes of beads that are impregnated with dyes that emit yellow fluorescence when excited by either a UV or a blue LED. These beads are very uniform; measured in a flow cytometer, the coefficient of variation (CV) of fluorescence is typically under 2%. CV’s under 4% possible with imaging systems by correcting for variations in illumination over the field of view (done with uranium glass, the fluorescence of which does not bleach or fade). There are millions of beads in a few drops. The LEDs in the existing illuminators can put out enough photons so that they will not be a major contribution to variance.