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Scan Mode Scanning Multi-Layer Regions (Image Stacking)
Scan Mode

Information in this article applies to:

  • uScope Navigator V4.5 and Later

Article ID: NSC1175 — Created: 1 Sep 2018 — Reviewed: 9 Sep 2019

Summary

uScope Navigator v4.5 adds support for multi-layer scanning (Z-stacking or image-stacking). This new scanning method requires some manual work to generate good quality images.

Details

Image-stacking is a microscope slide scanning technique that captures multiple images of each scan field at different focus positions. The images are subsequently combined (stitched) into whole scan images of each focus plane. This allows the end-user to review the specimen at different focus positions. Refer to the following article for information about the multi-layer image viewer.

Image-stacking takes significantly more time to scan and process images. Therefore, it should only be used when absolutely necessary.

Typical pathology specimens are very thin (4-8µm) and are easily scanned by a single focus layer. Each objective has a specific depth-of-field (the distance between the nearest and farthest features that are in focus without moving the objective) that is measures in microns (for low-power objectives) or sub-microns (for high-power objectives). This is why focus is more critical at objective power increases.

Thick sections (greater than 10µm thick) are likely to have important parts of the specimen that are several microns above or below each other and that, therefore, cannot all be in focus at the same time. This would be the case with insects or thick smears. These specimens are prime candidates for image-stacking.

Setup Multi-Layer Scanning

Select Multi-Layer Focus Method To begin, enter a job name and select a region of interest. Then, select the Multi-Layer focus method from the Scan Configuration window.

For a successful multi-layer scan, you must know several data points:

  • The spacing between layers in the scan. The depth-of-field of the objective helps determine the spacing (in steps).
  • The total depth of the specimen. This requires the following:
    • The focus position of the "tallest" feature in the specimen's region of interest.
    • The focus position of the "shortest" feature in the specimen's region of interest.
  • The number of layers to scan.
  • The focus position of the top, middle, or bottom of the image stack.
Objective Depth of Field Recommended
Spacing
(Steps per Layer)
10x ≈5.0 microns 25-100
20x ≈2.0 microns 6-15
40x ≈0.6 microns 3-8
60x ≈0.4 microns 2-5

Objective Depth of field

This table provides approximate depth-of-field and shows recommended spacing for each objective power. The indicated spacing (in steps) is conservative. You may be able to increase the spacing when scanning multiple layers.

As you can tell from the information in this table, smaller spacing and a thicker specimen both increase the number of layers and the time required to scan the region of interest.

Depth of the Specimen

One way to determine the depth of the specimen is to browse the region of interest, focus several fields, and record the focus position from the browse screen. You should record focus positions from the middle and all four corners of the region of interest at a minimum. Then, determine the maximum focus position (FMAX) and the minimum focus position (FMIN). You may wish to extend the minimum and maximum focus values to provide a buffer for the scan. Then, use the following formula to calculate the focus depth.

Focus Depth = Abs(FMAX - FMIN)

Enter Focus ParametersNumber of Layers

When configuring a multi-layer scan, you must enter the spacing and the number of layers to scan. Use the following formula to calculate the number of layers.

Number of Layers = Focus Depth ÷ Spacing

Focus Position

When configuring the multi-layer scan, you must also select the starting position relative to the stack (top, middle or bottom). You must set the initial focus to the top, middle, or bottom of the stack selected.

PositionTop = FMIN
PositionMiddle = (FMIN + FMAX) ÷ 2
PositionBottom = FMAX

Refer to the following article for more information on configuring the multi-layer scan.

Note
The region size and layer details appear in the top right corner of the uScope Control Panel screen as shown in the image above.

Select Focus/Illuminator and Start Scanning

Once the multi-layer parameters have been entered, click the Start Scan button.

At this point, you must set the relative focus position (top, middle, or bottom) depending on what you selected for the Start Position for the multi-layer scan; set the illuminator level; and start the scan.

  1. Create Whole Scan Image DialogSet the focus level to the appropriate calculated focus position from above.
  2. Set the illuminator level for the specimen.
  3. Slick the Start Scan button to begin scanning.

Create Whole Scan Images

When scanning completes, you may create a whole scan image from the image stacks. The image to the right shows the Create Whole Scan Image dialog for a multi-layer/EDF scan.

For more information about creating multi-layer whole scan images, refer to the following knowledge base article:

Note
Once you have a multi-layer scan, you may create an EDF (Extended Depth of Field) image. EDF is a technique that combines the best focused parts of each layer to form a new image that offers everything in focus.

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