Imaging problems
Gradient across sample or intensity difference left to right
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.
With multiple brains in particular you might see something as follows.
What is happening
In both the above caess the blade is 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 images, the blade is cutting ventral to dorsal. The blade is deeper on the right of the sample than the left, so when we image there is more tissue above the imaging plane on the left half of the image. The image looks a little dimmer on the right because the imaging plane is partly out of the brain. This often leads to more tiling artifacts on that side. In the following example, the problem is going the other way.
Temporary fix
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.
There is fluorophore cross-talk (bleed-through) between channels
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 some cross-talk between channels since the emission spectra overlap.
You can not solve this by altering the PMT gains. Decreasing laser power might help: certainly using more power than is necessary is not going to help. You can also try different wavelengths. For example, if GFP is strongly bleeding into tDomato at 920 nm, then try 800 nm. With careful power and wavelength choices, bleedthrough can be minimized. Should this not work, you will need to address the problem 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.
Optical planes are varying brightness and contrast
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.
For the solution see "Confirming the beam intensity with z-depth" on the Starting the acquisition page of the user guide.
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