Slide scanning workflow >

5. Scan settings

Overview

Enter more specific information about how you want to conduct the scan.

Note that additional information is needed for 3D slide scans, compared to 2D slide scans.

Procedure

Stack options [for 3D slide scans]

Desired height: Enter the thickness of the specimen in microns (or the portion of the specimen that you want to acquire)

Distance between planes: Enter the desired distance in microns between 2D image planes in the 3D image-stack.

Focal planes [3D slide scans]

The number of focal planes in the image stack that will be acquired is reported here. It is a function of the Stack options entered.

Stage settle delay

This option has no effect on 3D scans.

You may need to pause image acquisition after stage movements to allow time for vibration to subside.

  • We recommend setting the Stage settle delay to "zero" initially and conducting a sample acquire to evaluate if a pause is necessary (step 8. Sample acquire).

  • Examine the sample image and revisit this step to increase the Stage settle delay if the image is blurred, indicating that the stage was moving when the image was captured.

    • If there are artifacts with no Stage settle delay, a delay of 100 ms is usually sufficient.

    • With slower stages, a Stage settle delay of up to 1000 ms may be needed.

For fluorescence or other low light images that require integration by the frame grabber, enter a value larger than the time of the integration (as much as two times the integration time, but start with a number only slightly larger, and increase if you see text in some individual tiles). Neurolucida does not know how long the acquisition is, so be sure to allow time for the frame grabber integration.

Overlap / trim for images

Overlap percentage: Type in the desired amount; we recommend:

  • 10% when there are no or minor unwanted edge effects on images of each field of view (image tile)

  • 20–25% or more when there are significant unwanted edge effects, depending on the severity (size) of the unwanted edge effects

The display lists the number of pixels that will be trimmed or that represent the percent overlap (calculated from the Overlap percentage entered). These values represent the trim/overlap prior to stitching; the final values may vary somewhat from these estimates if you include the recommended Stitching XY option in Step 11. Start scanning.

Neurolucida assembles image tiles captured from each field of view into larger images/image stacks. First, the software arranges images according to the position of the microscope stage when the image was captured.

  • If you check the box to include the Stitching XY option in Step 11. Start scanning, Neurolucida fine-tunes the alignment by registering visible features in the individual image tiles; at least 10% overlap is required to facilitate this process.

    • Once the individual image tiles are aligned, the overlap is trimmed down the center. The amount trimmed from each image tile may be more or less than half the overlap, depending on how much the stitching process repositioned the image tiles.

    • Stitching adjusts for minor discrepancies in stage position that can be caused by factors including thermal drift, sample movement on the slide, stage movement inaccuracy, and gear backlash.

  • If you do not employ stitching, image tiles are aligned based only on the stage position when they were acquired. The pixels trimmed will match the values displayed in the workflow.

Blending

Blending is designed to minimize the appearance of tiling in your compiled image by averaging the pixel intensities where image tiles meet.

  1. Check the box to Enable blending

  2. Confirm or specify the Blend width in pixels; to change the blend width, enter a different number of pixels in the box.

Background color

Applicable only if the Scan type selected in step 1. Setup is Scan inside contour only.

  • Click to select white or black to be added the edges of the image.
  • Generally, white is used for brightfield imaging and black is used for fluorescence imaging.