Virtual Tissue (slide scanning): Acquisition settings

Grid

Use to define the area to be acquired as a set number of fields of view in a rectangular grid. This option requires that you be positioned at the top left corner of the region to be scanned; the current field of view is the first one scanned.

Test the settings over a 3x3 grid before performing the full acquisition to ensure that the background image is satisfactory, that the trim and blend are appropriate, and to determine whether a stage delay is needed. This will save you considerable time!

Contour

Use only closed contours.

• ( only inside ): Processes tiles included in the contour that was drawn. The image may have rough, jagged borders but will save on acquisition time and file size.
• ( all tiles ): A rectangle is generated that contains the entirety of the contour. All fields within this rectangle are scanned and included in the final Virtual Tissue output. The final image doesn't have rough edges but acquisition time and file size are increased.

Type

• 2D: Appropriate when only one image plane is desired.
• 3D: Appropriate when more than one image plane is needed to create an image stack. This is generally appropriate at higher magnifications, but can be used at lower magnification as well (3D is only available if you purchased the Virtual Tissue 3D module.)

Height of Virtual Tissue: Refers to the desired stack height. The values you choose will affect the processing time.

The number of planes in the stack is determined by the Stack Options.

• To modify the number of planes, modify the Stack Options' values.

The program automatically adds the top plane to the number of focal planes.

The total number of images to be acquired is dynamically displayed. This allows you to balance image data acquired with the time and disk space necessary to collect an image volume.

• Uncheck all boxes to rely solely on the focus map (to be set up in the next step).

Manually, every: Check if it is necessary to manually focus the tissue periodically throughout the scan.

• Enter the number of images between each manual focus site.
• The acquisition stops at the specified increments and Neurolucida prompts you to bring the tissue into focus before continuing.
• If this is being used simultaneously with a focus map, the remaining tiles on the triangulated map will be recalculated after each manual focus.

 

Automatically, every: This option requires special hardware. If this option is grayed out, the hardware is not installed or connected. To enable this option, see Autofocus in General Preferences: Lens.

Useful for stages that drift. Use manual focusing only if you need to repeat a completed scan that shows a significant loss of focus toward the bottom of the image to correct for drift.

Set Autofocus Range button: Check the Automatically box to enable the button; use to define a range (see Autofocus in General Preferences: Lens for details).

Minimum energy: 

Use the Get button to have Neurolucida determine the minimum value.

Energy is a measure of contrast in the image. In general, as the image comes into focus, the energy value increases, and the image with the highest energy corresponds to the best focus.

• If there is very little in the image (e.g., a hole in the tissue), the energy values tend to be low because there is not much contrast. For this reason, you may want to avoid that area as a focus location.
• If the autofocus is done in a region for which the best (highest energy) focused image has an energy value below the Minimum energy value that you defined, the software discards the autofocus result but maintains the Z value of the region until it can identify another region that has more to focus on.

If you are acquiring an image with brightfield illumination, the time delay is used to allow time for the stage in XY to stop moving and any vibration to die down.

For your test montage:

• Set this value to "zero" first
• Increase the value only if you see signs that the stage is still moving when the image is captured (e.g., blurred image).
  • If there are artifacts with a zero time delay, 100 ms is usually a sufficient delay.
  • With some of the slower stages (especially Prior stages), delays of up to 1000 ms may be needed.
  • If you are acquiring fluorescent 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). The software does not automatically know how long the acquisition is, so be sure to allow time for the frame grabber integration.

  This option has no effect on Virtual Tissue 3D acquisitions.

 

Options available for 2D and 3D:

Keep Image Open: Displays the image in the Image Organizer when the acquisition is complete for immediate inspection.

Multichannel Acquire: Uses the existing Multichannel acquire setup; adjust the Multichannel Acquire settings prior to performing a Virtual Tissue acquisition.

Compress Tile Files: Reduces the size of the image files.

• Use the scroll bar to adjust the compression.
• Generally a compression of 10:1 or 20:1 is adequate (meaning a compression of 20:1 will take a 100 MB image file and compress it to around 5 MB).

 

Options available for 3D only:

Remove Temp Files: Individual image files are saved in a folder until they are compiled into the final image montage at the end of the acquisition.

• Check Remove Temp Files to delete the individual files once the image has been compiled (this will free up disk space).

Postpone Compilation: Individual image files are acquired and saved in a designated folder, but there is no compilation performed.

• Uncheck Keep Image Open and Remove Temp Files to enable the Postpone Compilation option.
• Use the Virtual Tissue Compiler when you are ready to create the montage.

Capture Virtual Tissue: Enter the distance in microns if the image needs to be acquired below the focus map.

Useful if you want to create the focus map at the top of the tissue, but capture the image a set distance into the section thickness.

• When you designate a distance to acquire below the focus map, the first plane of the stack is acquired starting at that specified distance.
• If you enter “0”, acquisition will occur at the designated focus map locations, or at the specified top of stack.

Pixel Trim Images: Removes rows and columns of pixels from the edges of each field of view (image tile).

This option allows you to correct for spherical aberration or a video card that acquires with a black or white strip on any of the image edges.

Try a test acquisition with all of these values set to zero, and increase them if there is a problem in the final image.

1. Acquire a single image
2. Zoom in on the top left and bottom right corners. Any rows or columns of bad pixels are immediately evident.
3. Set the trim values to remove the unwanted pixels.
   • For low magnifications, 0-10 pixels is a suitable range.
   • For higher magnifications, larger trims (25-50 pixels) may be appropriate.

• If lighting is very uneven across the field of view, large numbers of pixels may need to be removed to account for unevenly lit portions of the image
  • Try to correct lighting before resorting to trimming.
• When many pixels are trimmed, the fields of view in the resulting virtual slide are made smaller, and smaller stage movements are made accordingly.
• High values for trimming will slightly increase your acquisition time, since more acquisition sites are required.

Seam Blending: Blends the edges from each field of view smoothly.

• Use the 3x3 test first to see if your image looks acceptable without blending (try to avoid blending as it involves a slight loss of image detail).
• Select the number of pixels to blend at each seam. Generally, 5-10 pixels is sufficient.

Background Color: Use if the image scan selected is Contour (only inside).

• The color (White or Black) will appear around the edges of the image (white in this example: ).
• Generally, white is used for brightfield imaging while black is used for fluorescent imaging.