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Hi Tim,
Just put some green PS Speck beads on a slide and image at 800 nm, when the image is not too far off the expected values, your system should be fine. If you have a convallaria test sample, this should give a bright signal at 800 nm with just a few mW and low detector gain. How deep into the tissue are you trying to image? It might be good to underfill the back aperture to gain signal at the expense of z-resolution.
Best wishes
Andreas
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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