Setting the gain for a scan

It's one thing to have been shown which knobs to turn to get an image and another to get some understanding of what the knobs are actually doing and how to adjust a number of free parameters during scanning. Basically, the image you will get in a given "optical situation" (i.e. with a given prep and a given objective) depends on three parameters: Laser intensity, detector gain, and pinhole size.

1. Laser intensity:

Just to clarify this: There are 2 ways of regulating it. The knob on the control panel under the keys regulates the voltage that is applied to the gas. Don't touch that, Ed has set it to some standard and safe value. In the software, you find sliders from 0-100% for every line. These control an acousto-optical transmission filter (AOTF) that attenuates the intensity of the laser in a wavelength-specific way. In theory, you want the laser pretty damn bright, but of course you don't want to bleach your fluorophores too much.

2. Detector gain:

The detectors (photo-multipliers) are basically cathode-arrays that amplify the light signal. Again, it would be nice to have them as sensitive as possible. The problem is that they are subject to thermic noise. That means, at too high a voltage, they count photons that are not really there and the image gets noisy. As a rule of thumb, don't set them to more than 500.

3. The pinhole:

The pinhole diameter determines the "confocality" of your image. The smaller the pinhole, the narrower the focal plain and the better your axial (z-) resolution. However, the smaller the pinhole, the less signal goes through. I recommend to leave the pinhole alone. By default, the scan software sets it to a specific value for each objective. It's called "1 airy" and refers to the "airy disc", which is the diameter of the first maximum of the theoretical point spread function (never mind, if you really want to know, refer to the Pawley book).

In summary, setting the parameters is a tradeoff between different things. Here is my default way of doing it:

1. Make sure the pinhole size is at "1 airy"

2. Set the detector gain to 500.

3. Use the laser intensity to get a bright enough image. If you use the "glowOver" colormap in the scan software, the brightest value (255)* shows in blue. Set the laser intensity so that blue just starts to show up in the brighter parts of your staining.

*the default setting for pixel depth (how many gray values there are to code for intensity) is 8-bit (0-255). You may find that this is not always sufficient for the intensity differences found in the kinds of stainings we produce and you find that you have to overamplify the bright parts in order to see the faint parts. There is the option to use 12-bit coding (0-4095). Hower, I don't recommend it. Your image stacks get really large and not all image softwares can read 12-bit.