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.

Effect of a tilted blade

With multiple brains in particular you might see something as follows.

Same thing showing the BakingTray preview window and also the on-line preview, which is multi-channel and has tile illumination artifacts corrected.

What is happening

In both cases above, the blade is not parallel 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 more severe and is going the other way (blade deeper on the left).

The blade is tilted such that it is cutting deeper into the block on the left. Note there are also perfusion-related artifacts on the bottom right brain.

How to fix this

Did you forget to push down on the blade holder when tightening it? Loosen the thumb-screw, push down, and tighten it. Cut and check the tilt. If this does not fix the problem, you could try changing the blade. If this also does not fix the problem, you can simply move the objective down a bit. This is a reasonable temporary fix if the tilt is not severe. If the tilt is severe and the samples are important you will need to alter the blade angle. This is a little tricky: if you are not aware how to do it you should get help.

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.

Emission spectra of eBFP, eGFP, and tdTomato

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.

Effect of increasing laser power too much with depth

For the solution see "Confirming the beam intensity with z-depth" on the Starting the acquisition page of the user guide.

White matter in deeper optical planes always look dimmer

White matter will always look dimmer in deeper depths: there is nothing you can about this other than cut thinner and start nearer the surface. However, you also probably do not need to worry about the images not looking identical in depth. We correct for this in the downsampled image stacks generated by StitchIt in case it might influences image registration. This is shown below:

Orig downsampled stacks

Corrected downsampled stacks

The full-sized stitched images are currently not corrected, but code exists to do this if you need it. So far nobody has asked for this.

High frequency laser fluctuations (laser noise)

Multi-photon lasers deliver their light in short, very intense, intense pulses. This is necessary for the two photon effect, which occurs when two long-wavelength photons superimpose in time and space to behave as a single short-wavelength photon. Through this mechanism 920 nm IR light can take the place of blue light to excite GFP. The process of generating the pulses is very sensitive and sometimes lasers will rapidly switch between pulsing and not pulsing or deliver poor pulses. The resulting images show high-frequency stripes and tend to look dimmer than normal. An example of this is reproduced below. If you notice this happening, you can often side-step the problem by changing wavelength by a few nm. Perhaps 5 to 15 nm is needed. These small changes in wavelength tend to have minimal impact on fluorescence signals. Sometimes the issue only manifests after the laser has been on for two or three hours. Always report such incidents to the system power user.

A detail of an image taken in the presence of laser noise. This image is taken on a 4 kHz resonant scanner with 4 frames of averaging.

A detail of a normal image from the same sample showing no laser noise.