OD meausured via scattering as opposed to absorbance

[anon1234567]

Hi there, I know the Chi.bio can measure OD via absorbance and scattering. I want to grow cyanobacteria and as you are well aware, the 650nm laser is not sufficient due to the absorbance of photosynthetic organisms. I can’t figure out how to switch the OD measurement mode from absorbance to scattering and would appreciate some help

[harrison]

Hello,

To see how to do this check the function MeasureOD in app.py. In short what you need to do is change the value stored in sysData[M][‘OD’][‘device’] (by default this is ‘LASER650’ to something else (e.g. ‘LEDF’)) which will then mean the calibration and measurement OD functions will use this light source instead. There are different ways of calibrating this, depending on what level of precision you are hoping to achieve. Namely, you can calibrate just the zero-light level as usually done with the laser measurements. Or, you can go a step further and try to calibrate out inter-device variability, which you can see (some) code for in there already, and brief description in the Chi.Bio paper’s supplementary.

Harrison

[anon1234567]

Hi Harrison, thanks for the response. So if I understand correctly, I would change the ‘LASER650’ to one of the LED’s (eg. LED623nm). Is that all that is required as in the supplementary paper you also mention different correlations used for absorbance as opposed to scattering. Would I need to further modify the code or is what I stated above sufficient for measuring OD via scattering?

[anon1234567]

Hi, just a follow up comment. Looking at the code I think you have already put an else if function for LEDA and LEDF in the measureOD function in App.py, with the corresponding OD calibration constants. Am I understanding this correctly and if so, would just modifying the first two ‘LASER650’ lines to ‘LEDA’or ‘LEDF’ be sufficient to measure OD via scattering?

[harrison]

Hello,
To change to LEDA or something else, you need to change the value I mentioned in sysData, which is defined at the top of the code.
You could then change one of those else if statements to catch whichever LED you choose (i.e. swap out LEDA for LEDD, assuming you want to use LEDD).
Then it should work as you hope, though it won’t be calibrated to align exactly with whatever benchtop spectrophotometer or plate reader you use. In practice spectrophotometers and plate readers can vary dramatically between manufacturers and devices, often by a factor of two or three!
So, if you want the Chi.Bio to match whatever other measurement device YOU have, then you would additionally need to do calibration by measuring a bunch of solutions on both the Chi.Bios and also whatever other device you want their measurement to replicate – as described in the paper Supplementary.

[anon1234567]

Hi Harrison, thanks for that, it is working now ! I am measuring with the OD LEDA (395nm). With regards to a calibration curve my thought process is to grow the cyano cells to a very high OD (eg 15) and then measure the OD with the Chi bios and the bench top spectrometer at 730nm. I will then dilute multiple times while measuring in the same process, to form a curve. Does that seem like a good way to calibrate the online OD measurements with the bench spectrometer?

[harrison]

I think that is reasonable plan and aught to achieve what you are aiming for!

Keep in mind that both devices (Chi.Bio and the spectrometer) may be inaccurate at high ODs since it enters the “multiple scattering” regime. For example, this is why generally you need to dilute samples before putting them into the spectrometer when at high ODs…

[Noelia]

Hi both,

I was able to do what is recommended here. I have the calibration curve for different ODs measured both in the ChiBio and spectrophotometer. Is this calibration curve external? Or can I add it somehow in the code so the OD is automatically calculated in the ChiBio?

Thanks!

Noelia

[harrison]

It is defined in the measureOD functions. You can define it in there, or if you wished do some external post-experiment recalibration to the OD – but of course this would mean your in-experiment set point OD would be different.

[Author]

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