Re. Spectral Detector Calibration

[Glyn Nelson]

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Hi Claire,

I would echo Sylvie’s comments- do you see a difference in results between the same fluorophores before and after the service with the two correct reference spectra sets?  My feeling is you shouldn’t, and it should correct for the weightings.

To answer your specific questions, I am well aware they need recalibrating  now and again- I had problems with one of mine and had to get an engineer to recalibrate it.  And yes, I inform users that they should recapture reference spectra.  I think as part of the PM service, they recalibrate both the grating positions and normalisation of the detectors to one another.  I have seen differences between detectors in the array even after such a service, and in a way, I prefer the poor man’s version, using one detector and a sliding dichroic.  So, I think if you see differences before and after a service, it is more likely to be the normalisation of the PMTs rather than the spectral positioning (if you use new reference spectra).

Regarding the QC of the system for reproducibility, this is far trickier- I can only think that taking a well defined and stable spectra to show that the windows are where they should be is about all you can do, as several others have suggested using an acetate slide or stable excitation source.

A word of warning if your users are trying to quantify intensities from such data though- the algorithm they use (or at least, the ones I read up on when the LSM meta heads first came out- I think it is still the same) essentially splits the data in each pixel to the reference spectra with weighting.    1., I struggle to see how this can ever be as quantifiable as simple intensity data from a PMT (yes, I know that is another argument, but this is adding another layer of variables on top!), and 2. The weighting is always going to be different between samples if they have been unmixed with different numbers of spectra or with/without residuals, so this should always be consistent too.

Glyn.