How were the laser calibration factors for OD determined

[jeff8838]

Hi,

How were the laser calibration factors for OD determined?

In app.py, calibrateOD() and measureOD() grab values from a and b:

a = sysData[M][‘OD0’][‘LASERa’]
b = sysData[M][‘OD0’][‘LASERb’]

From the sysData dictionary, it looks like a = 0.226 and b = 1.833.

How would I go about determining these values if I wanted to use another light source?

Thanks for your help!

[harrison]

They were done by calibrating against a spectrophotometer in our old lab. If you wish to see how it is done, check the supplementary material of the Chi.Bio paper in PLoS biology which shows this raw data + the fitting used. In that case various standard solutions were used in the calibration, but you could also use whatever cell type you are using in your research to make sure it matches up perfectly across your different instruments.

[harrison]

They were done by calibrating against a spectrophotometer in our old lab. If you wish to see how it is done, check the supplementary material of the Chi.Bio paper in PLoS biology which shows this raw data + the fitting used. In that case various standard solutions were used in the calibration, but you could also use whatever cell type you are using in your research to make sure it matches up perfectly across your different instruments.

[jeff8838]

Thank you – I found the information I was looking for in the Chi.Bio paper.

[jeff8838]

Hey there,

A follow-up question: is the “a” variable (sysData[M][‘OD0’][‘LASERa’]) similar to the 0.3974 term in front of ODi in equation (S2) of the supplementary paper, and is the “b” variable (sysData[M][‘OD0’][‘LASERb’]) the same as the 1.374 term in front of ODi^2 in equation (S2)?

[jeff8838]

Also, how did you derive the following equation in the code?
Do you use the following equations: OD0 = ODRaw/ODActual, and ODi = log10(OD0/ODRaw), and the general quadratic equation for ax^2 + bx + c = 0?

raw=((ODActual/a + (b/(2*a))**2)**0.5) – (b/(2*a)) #THis is performing the inverse function of the quadratic OD calibration.
OD0=(10.0**raw)*ODRaw

[jeff8838]

Okay scratch that – I figured out how things were computed 🙂

[Noelia]

Hi there,

To get the raw measurements in the ChiBio do I need to set LEDFa=1? (I’m doing it with scattered light). I’m doing this to calculate LEDFa with my cells and media through a curve calibration and I don’t want the default calibration to interefere.
Same for absorbance, should I set Lasera / Laserb differently?

Thanks,

Noelia

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