Public Lab Research note

Calibrating a Microscope

by mathew | August 23, 2016 17:19 23 Aug 17:19 | #13391 | #13391

image credit wikimedia

What I want to do

I have an openflexure microscope and I want to calibrate it. I've acquired two tools to do so, a stage micrometer, and 2.07μm Polystyrene Latex (PSL) Spheres by Ladd Research. I have previously documented the method for calibration with the stage micrometer that i learned from @AmberWise and Andrew Maselli at Chicago State, and recently replicated it with my OpenFlexure microscope.

But in order to check the particle sizing methods @SimonPyle has automated for use in dust sensing, I would like to compare particles of a known size to the measurements the analysis produces.

Experimental Goals

My goal is to understand whether a stage micrometer calibration is sufficient to accurately size small particles, since the stage micrometer is reusable and fairly affordable (less than $100). After analysis, I should be able to compare the average particle size of the particles (2.07μm) to the measured particles size.

This will lay the groundwork for running (passive particle monitor samples)[] here in support of our development goals.

Proposed Process

I'm going to prepare a slide with the PSL spheres, take a calibration image of the stage micrometer, and then take 40 images of the prepared slide with the microscope. then run it through @simonpyle's ImageJ macro, followed by the analysis spreadsheet @AmberWise and I put together.

Next Steps

time to do this!

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Here are some image sets including a flat field calibration and scale from the PSL imaging.



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Saw this during the air pollution open call today, It looks really nice! any reason you used ImageJ and not Python + OpenCV?

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