Mounting medium for multiphoton microscopy

Hello,
We did some experiment with mounting medium and some mounting medium “burn” the slice sample (I see some sort of black spot on the slice and more i stay without movement more they become bigger) during the acquisition with a Nikon A1RMP+ multiphoton microscope.
Now we image without mounting medium but I want to image the sample with fluorescent labelling on different microscope.

Do you have advice?

Best,

Alex.h - Hmmm. I also have burned my mounted slide on a multiphoton. There isn’t much heat dissipation in glass and hardened mounting medium, this is the big issue. I’m sure you already tried the imaging tricks - keep power low, increase gain, do plenty of averaging … I wonder if wet-mount mediums might tolerate the localized IR heating a bit better…Have you tried imaging the slice first with multiphoton, then mount the slice for regular single photon? Remember also that mounting is reversible once its on the slide, so in theory you could mount and image, then reverse the mounting by incubating in a humid chamber, perhaps rinse the slice, then image with the A1RMP…(I’m brain storming).

Related, I’m wondering if your objective on the A1RMP is coverslip corrected - if not, you might get a better image with water (can therefore use less power to get the same signal), no glass. Also, a hint in general Genteal eye gel works very well for multiphoton microscopy, its thick doesn’t evaporate, and has a refractive index like water/tissue.

Hello,
Thank you for your advice. I will try with liquid mounting medium and with Genteal eye gel.

Best,

personally, for tissue section imaging, I find that wet mounts under a coverslip work much better for multiphoton imaging. I’ve been able to get away with pretty high powers (>10mW) while retaining tissue integrity.

It’s not great for long term storage though. is that a concern?

-Wilson