Step 1: Setting imaging parameters

Select a directory to which data will be saved

Hit the "Dir" button to select a directory where data will be saved. Do not use spaces in the directory name. Your current working directory in MATLAB will be unchanged. The Sample ID (see below) is automatically set to be the same as the directory name, but this can be changed in the next step.

Edit the recipe

The "recipe" describes the acquisition parameters, edit to your needs. Here we have changed:

  • Tile Size describes the imaging resolution.

  • Scan Mode describes how the tiled acquisition will be performed. In "Manual ROI" mode you draw a ROI over the sample and this same area is imaged in all sections. In "auto-ROI" the sample is automatically identified and the ROI re-calculated after each section.

  • Sample ID is a string that defines the sample name and is used for file naming. A default is provided if you enter nothing.

  • Slice Thickness is the physical section thickness in mm. i.e. how much is sliced off the block each time. Here this is 40 microns, which is a reasonable compromise value.

  • Num Optical Planes defines the number of optical planes within each section. Here this is 2, meaning 2 equally spaced optical planes imaged within each physical section. The separation between these is listed in the text above the recipe: here 20 microns.

  • Num Sections is the number of sections we will cut over the whole acquisition. Here this is 275 sections, with equates to 11.0 mm of imaged tissue (see text above recipe entry boxes). This is the number of sections multiplied by the thickness of each section. The number of allowed sections is automatically capped based upon the current remaining travel range of the z-stage.

  • Cut speed defines how fast the blade travels through the sample in mm/s

You may image multiple samples at once, but all must share the same acquisition parameters.

The parameters at the bottom are advanced settings that you likely do not need to ever alter.

Select the image resolution

The resolution of the acquisition determines the number of microns per pixel. You should use the pop-up menu shown above to select from one of the pre-defined resolution settings. It is important to choose the correct resolution for addressing your question. Usually you need a lower resolution than you think: ask if you are unsure about this! Once you have done this, you will see that the image size in ScanImage and Tile Size in BakingTray have updated:

Frame averaging

If you expect the SNR to be low, you might choose to average frames to improve image quality. For resonant scanning, this is achieved by altering the Frame Average number in the ScanImage Image Controls window (below). You may alter this number once the acquisition started but you have to wait until the sample is being sliced to do so. For linear scanning you should instead alter the pixel dwell time in the ScanImage Configuration Controls.

Do not become too preoccupied with the image quality of the autofluorescence. The main use of the autofluorescence signal is for registering to a standard atlas and this is achieved using a downsampled image stack, which is heavily averaged when it is constructed. The intensity of the fluorophore signal is substantially higher than the autofluorescence, so in most cases no averaging is needed for downstream analyses to be successful. For more information on frame averaging and examples of how effective it is see Choosing a Resolution.

As a rule of thumb, a microscope with an >1 mm FOV can image six brains in under 24 hours at a resolution suitable for tracing bulk projections and electrode tracks. It will image four brains at cell counting resolution in under 24 hours, assuming no averaging. Avoid averaging with 4 cellfinder brains or imaging >4 cellfinder brains at once. See Choosing a Resolution or ask for more information.

The ScanImage CHANNELS window

Select which channels are to be saved using the "Save" checkboxes in the ScanImage Channels window. Select which channels are to be displayed on-screen during acquisition using the "Display" checkboxes. These settings can not be changed once acquisition commences. If you selected multiple channels to display, all but one will be automatically disabled after the first section is complete for performance reasons.

Note that a friendly name for each channel is provided in the PMT GUI, as shown below. In the case of the example system shown, Channel 2 is "green".

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