Step 1: Setting imaging parameters
If necessary, start ScanImage and BakingTray. The instructions over the following pages are summarized as a checklist.
Start a new sample
Select Sample -> New from the menu. This will reset the recipe and turn on the resonant scanner (if present).
Turn on the laser
Hit the "Laser" button in the main view then press "Turn On" on the laser window. After hitting this button the text switches to "Turn Off", as shown above. The power will increase slowly and eventually the modelock indicator will turn from red to green.
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. Here this is 40 microns.
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 can change settings in ScanImage, but in order to maintain good calibration between the image size and the actual number of microns per pixel you should use the pop-up menu shown above. Instructions for setting up this menu item if it doesn't exist are here.
Once you have done this, you will see that the image size in ScanImage and Tile Size in BakingTray have updated:
Advanced usage: non-standard image resolutions
The tile size and image resolution shown in the above status text and in ScanImage are the values that will be used during acquisition. If you set the tile size with the pop-up menu then subsequently modify it manually in ScanImage, the value in the pop-up menu is no longer relevant and will be ignored. The label text "Tile Size" will turn red when this happens as a warning, although you may proceed with the acquisition. The label text will also turn red if the zoom setting in ScanImage is changed, since then the microns per pixel value in the pop-up menu is no longer correct.
BakingTray will honor the values you have chosen but you may well need to tweak stitching parameters post-hoc to get nice stitching. It is, therefore, not recommended you over-ride the built-in pixel size options
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".
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.
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