Problems and solutions
You see this odd gradient in your images and the gradient is worse in upper optical planes. It may be disappear completely in deeper optical planes.
Effect of a tilted blade
What is happening
The issue stems from the blade not being flat with respect to the Y stage. The Y stage is that which moves the sample along the direction parallel to the blade. In the above image, the blade is cutting ventral to dorsal. The blade is deeper on the right of the sample than the left. The image looks a little dimmer on the right because the imaging plane is partly out of the brain.
Start the first plane deeper into the sample to avoid the problem area.
Full solution for super-users
Correct the blade angle by doing a roll adjustment. i.e. don't alter the pitch of the blade (it's angle of attack into the tissue). The blade angle can be corrected by imaging a fluorescent block of agar (dope it with marker pen fluid) and tweak the angle until two points about 15 mm apart are within 10 microns of each other after cutting.
For speed reasons BakingTray acquires all channels simultaneously at a single laser wavelength. Since 2-photon excitation spectra are broad, it is even possible to acquire red, green, and blue fluorophores at single excitation wavelength (780 nm). There will inevitably be cross-talk between channels since the emission spectra overlap.
Emission spectra of eBFP, eGFP, and tdTomato
You can not solve this by altering the PMT gains or laser power. Instead you need to address the issue at the analysis stage. This could involve simply overlaying multiple channels to identify which fluorophores are present where. Alternatively, you might want to consider some sort of unmixing strategy.
Deeper optical planes will naturally be dimmer due to scattering of excitation an emission light. Water immersion objectives aren't corrected for the refractive index of fixed tissue, so imaging deeper will produce more blurry images due to spherical aberration. Issues relating to section thickness are discussed on the Choosing a resolution page. If, however, you see an obvious increase or decrease in overall signal intensity with depth then you likely did not set up the sample properly.
The image below shows a four brain acquisition with four optical planes spaced 12 microns apart. The brightness increases with depth because the exponential depth constant in ScanImage was set incorrectly. The stripe pattern over the image is electrical noise that is noticeable because this sample was acquired at a low laser power.
Effect of increasing laser power too much with depth
If you are having problems with cutting the first thing to do is consider the blade you are using. Particularly if you are using a thinner blade, consider changing to Campden Instruments stainless steel blades, which are verified to work well. These do not rust and are very rigid. Buy in bulk: they are cheaper that way. Expect to use a new blade for each sample but in practice you can image multiple sessions with the same blade if you wish.
Next consider your cutting parameters. In general cutting 40 micron cuts at 0.5 mm/s should work. You might need to cut a little slower in some samples: perhaps down to 0.35 mm/s. Sections thinner than 40 microns might not sink easily and remain floating on the surface or not will away from the agar.
Alternate thick and thin cuts
Vibratomes have a tendency to enter a feedback loop where they cut alternate thick and thin sections. Using the Auto-Trim feature or otherwise slowly stepping down to the final cutting thickness should help reduce the chance of this happening. Sometimes some samples are tricky and this doesn't help. If so, try cutting slower: 0.35 mm/s. If that doesn't help, then take a single section about 50% thicker than go back down to your target thickness. If this also doesn't help, consider imaging with sections 10 to 15 microns thicker.
BakingTray controls the laser so that acquisition can be automatically halted if modelock is lost. There are laser control classes for MaiTai and Chameleon lasers. The laser can be controlled via the the "Laser" button in the main GUI.
Sometimes initial connection to the laser fails. If this happens try the following. Firstly, attempt to open the laser shutter because sometimes the laser is in fact connected:
hBT.laser.openShutterIf that fails (it returns
0) then you will need to try disconnecting and reconnecting to the laser with
hBT.renewLaserConnection. You can also try this manually:
hBT.laser = maitai('COM1'); % assuming you have a MaiTai on COM1
hBT.laser.parent = hBT;