In an exceptionally useful paper, Lees et al. provide a set of protocols for checking the performance of your multiphoton microscope: Standardized measurements for monitoring and comparing multiphoton microscope systems (link to preprint).
The paper covers the following procedures:
- Measuring laser power at the sample
- Measuring FOV size
- Assessing FOV homogeneity
- Measuring spatial resolution
- Optimizing group delay dispersion
- Measuring PMT performance
- Estimating absolute magnitudes of fluorescence signals
Check it out – it is definitely one of those reference papers that are great to have around in the lab space!
A blog about neurophysiology 
Nice paper, so my thought is. Should we be including measurements of the characteristics of our setups when publishing any information on new genetically encoded or synthetic optical sensors.
And from such measurements could a tool be made to predict from the raw data what you could expect from the same sensor on your old 2P system with multi-alkali detectors perhaps (with similar standardised measurements)? Compared to the ofter highly optimized systems used when characterising sensors.
That’s a good question! I believe that we always have to strike a balance to only include measurements of the setup characteristics that are to some extent relevant for your study. For example, if the sensor you study is very dim and you need to optimize the PSF and the pulse dispersion a lot before you get good signals, then I would report it. But it is probably not possible or useful to report all these metrics anytime when a 2P microscope is used.
For your second question: it would be really nice to predict from the raw data what to expect from the same sensor with a different 2P system. I know from my own experience how difficult it is to track down different performances between different 2P systems. To give an example, pulse dispersion of the laser pulse might be different across setups because one of the setups includes a defective component (e.g. a mirror), resulting in a difference of fluorescence yield. Such an effect would be very difficult to capture, even with the very good set of metrics presented in this paper. So, in general, I like the idea of predicting fluorscence yield (and the paper nicely gives instructions on how to estimate the photon count), but there will be always an uncertainty unless you invest a lot of time in detailed comparisons.
And I agree that this situation is not great because it prevents easy comparisons of absolute values across studies, especially when testing and characterizing new and rather dim sensors…