Calibrating LED intensities

[saurabhmalani]

Hi there!

My lab does optogenetics with EL222, and so to maintain consistency between reactors I wanted to calibrate the intensities of the Blue (457nm) LED in all the reactors we have. I was trying to do so by setting the intensity of the LED to various values between 0.001 and 1 and measuring the output from the AS7341 spectrophotometer within the Chi Bio. I believe this is a similar procedure to the ‘Characterize’ function built within app.py

For our set of 8 reactors, this approach seemed to work well for 7 of those, but 1 reactor seems to be significantly off. Setting the 457nm LED to 0.1, 7 of them measure between 20000 to 27000 in the 440nm channel, but one measures about 45000. The variation in the 7 I could understand as variations between LEDs, but the 1 anomaly is definitely off. Setting the anomalous reactor LED to 0.04 gives a 440nm spectrophotometer reading of 23000, which would suggest its at least in the same ballpark as the other reactors, but visually it is very evidently dimmer than the others.

This suggests that there is possible significant variation, not just in the LEDs but also in the AS7341 chip, and maybe this anomalous reactor has some other electronics variations that makes this variation particularly bad.

Do you have any suggestions for how I can simultaneously address the variations in the photodiodes AND the LEDs to be able to calibrate the LED intensities? I saw from your supplementary that you deal with this variation in fluorescence readouts by taking the ratio of the desired channel with the clear baseband to cancel out both of these variations, and was wondering if you had any intuition for how I can do something similar to cancel out the AS7341 variations but maintain the LED variations so I can use it to tune the intensities of the LEDs accurately.

Thanks!
Saurabh

[harrison]

Hello Saurabh
I guess there are a few challenges
The first is how you measure the LED intensity with the spectrometer. In practice the spectrometer is at 90 degrees to the LED path so you only get a particularly good signal if there is something in the way (i.e. a test tube with media/cells) to scatter light into the spectrometer. Otherwise, what you are measuring is somehow the random internal reflections in the device which may be very sensitive to geometry. What I’d reocmmend is making some cloudy solution as your standard “scatterer” and then using this in all reactors.

Second, yes, there are sources of variability in both the LED output and the spectrometer. Probably the largest amount of this comes from small changes in alignment made during assembly when the lens is glued to the LED and the spectrometer is soldered in place. There is also variability in the individual LED emitters and spectrometer, which are off the shelf parts so there is nothing we can really do about this. We try tocalibrate this variation out for fluorescence measurements by doing the ratiometric measurement as you stated, but in your case you need a constant ABSOLUTE amount of light. I’d start by doing the above (standard scattering solution) and seeing how different it is, but if the answer is still “not good enough” then ultimately what you may need is to buy an off-the-shelf light meter that can be inserted into the top of the device so you can have a consistent measurement across reactors. This way you will know exactly the light output and have the same sensor for each (i.e. not using potentially variable AS7341 chips) to have the best possible calibration. You can get cheap light meters for <100 USD usually which are probably more than good enough to do the trick.

[saurabhmalani]

I see, you are right the 90 degree angle does add some concerns. In your paper, in the supplementary Figure S13 (Image Link: https://ibb.co/bJZptgr), if you still recall haha, how did you get that data? Did you use a ‘scattering’ solution of sorts like you suggest or was it just the naive readout of an empty reactor? If this data was collected from just an empty reactor, perhaps that may indeed be sufficient enough to give a decent calibration, though you’re right that using a scattering solution would likely be more rigorous.

[harrison]

IIRC I made some McFarland standards with medium ODs (i.e. 0.5 or something) and use those as a proxy for cells in media to do the scattering.
http://www.dalynn.com/dyn/ck_assets/files/tech/TM53.pdf
Another easy standard-ish solution to make is mixing small amounts of condensed milk with water.

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