Manual ROI acquisitions

Introduction

The manual ROI acquisition mode is a fallback mode for situations where the auto-ROI is not appropriate:

• You wish to image a rectangular sub-region rather than the whole sample.

• The acquisition has to be set up without the sample being visible. e.g. you have to have the entire organ and can not afford to trim anything. Note, however, that this situation makes set up more tricky as you can not estimate where the imaging surface lies.

• You have reason to believe the auto-ROI might struggle with your sample (e.g. it has very low SNR or tends to be extensively occluded by membranes).

If you consistently encounter minor problem with the auto-ROI, these can generally be corrected by altering how you set up or mount your samples. It is worth figuring out issues with the auto-ROI rather than defaulting to manual mode as a solution. It takes quite a lot of effort to get nice tight ROIs in manual mode and if you are not experienced at this you will either end up clipping tissue or you will create huge ROIs that image a large quantity of empty space.

As with the auto-ROI, you will draw a ROI around the area you intend to image and take a preview scan. The challenge with the manual ROI mode is that you have to ensure the ROI is large enough to capture the whole sample, which will likely grow in the X/Y plane as you cut down. You may also have to take into account tilt in sample mounting. You don't want a ROI that is excessively large as this will substantially increase the imaging time.

Instructions

Using the slide schematic as a guide, press the ROI button to draw a box around the area you expect the sample to lie. You can now drag this box to translate it and pull on an edge to re-size it. If you resize, it will snap to the nearest whole tile. Once you move or re-size the box, its size in tiles is shown at its mid-point. Double-click to accept the ROI.

Reposition and re-draw the ROI if needed.

Re-take the preview scan to confirm you selection.

When the sample is framed to your satisfaction, you can move on. Remember to take into account the changing size of the sample at later sections and also any tilt in the mounting of the sample that might cause it to appear to translate across the imaged field as cutting progresses.

Hints for getting tight ROIs

• Know the size of your samples. e.g. mouse brains that are not tilted along the D/V axis will comfortably fit into a ROI that is 9 mm by 11.5 mm. There is no point making your ROI larger, therefore, especially along the lateral direction where tilt is less of a problem.

• Set the laser to visible wavelength and use it as a GUI by scanning or using "point" mode at low power.

• Know your tile size. Use the tiles as a scale bar to assess how much a border your need to add if the sample is tilted. e.g. Mount brains with the ventral surface facing the blade as tilt along the D/V axis is the most problematic. Standing in front of the microscope you can estimate how much the brain will extend beyond what is currently visible in the preview window. Based on the tile size you can the estimate how much to enlarge the ROI and in what direction.

• Use the StitchIt Sample Splitter to crop non-imaged tissue at the end of the acquisition.