An easy check is to measure the power at the sample plane. You might have unexpected losses in the system that are reducing power at the sample.
For video rate, you also need much more power than usual since your dwell time is so short. If you are basing your power assumptions on galvo-galvo systems you will be drastically underpowered. You might just need to increase your laser power. 50mW out of the objective does sound reasonable though. How are you measuring output?
Another thing to check is the control voltages on your PMT gain to make sure they are operating where they should.
Finally, triple check your filters to verify they are correct and in good condition.
Craig
***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****Hi Tim,Which Mai Tai are you using and what is the center wavelength you've tuned it to? I think there are some fairly short pulse length variants of that laser which would benefit from pulse compression, but the normal ~100 fs variant is probably ok with it if the optics in your system are dispersion optimized (e.g. not too many achromats, etc).Do you have a sense if the optical design is reasonably good? Its usually pretty easy to spot problems like astigmatic scanning just by scanning a uniform fluorescent solution, but things like uncorrected spherical or chromatic aberration can be maddeningly difficult to diagnose without careful PSF measurements.MikeOn Wed, Sep 28, 2016 at 11:28 PM, Tim <[hidden email]> wrote:*****
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Hi, I hope the mailing list doesn't mind being bothered with another question. I am currently setting up a new custom resonant scanning two photon system (old Spectra-Physics Mai Tai, 8kHz resonant scanners from CamTech and new H10770B-40 PMT's from Hamamatsu). The problem I'm having is that when I attempt to do in vivo calcium imaging (GCAMP6s), it seems that my signal is too weak / SNR is too low when I try to keep the laser power after the objective below what I understand to be safe levels (< 50 mW). I have checked the alignment of the system multiple times including:
1) Beam is collimated and centered prior to Galvo's
1) Beam is centered and collimated after the beam expander following the Galvo's
2) Beam is centered at the back aperture of the objective
3) PMT collection lens position is adjusted for maximum PMT signal
The entire system is inside a cage system so alignment should be very close to begin with. Most parameters such as diameter of beam at back aperture of objective I have taken from what others have used in literature (18.5 mm for 20x Olympus objective)
I understand there could be many factors that contribute to this problem (such as GCAMP expression, cranial window quality on the sample side) although I have tested on multiple mice.
I guess I'm wondering if there are tests people use for troubleshooting a two photon microscope with lower than expected signal that are beyond the kind of alignment tests I mentioned above. Are there performance standards I can use (ex: for a given laser power after the objective and a given sample, one should be able to obtain a certain SNR) so that I have some idea how optimized my system currently is?
One thing we don't have in our setup is any kind of dispersion compensation. Could this alone be enough to allow us to image with sufficient SNR at lower powers?
Is it possible that the beam quality of the laser could result in low image SNR (aside from dispersion effects) and would it be something we could check?
Any help would be greatly appreciated,
Tim
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