Slide scanning workflow >

11. Start scanning

Overview

Choose options for acquiring and compiling images of your specimen and launch the slide scan.

The Scan Summary section of the workflow window lists information about the slide-acquisition settings and estimated disk space that will be required.

Procedure

1. If you did not create a focus map in the previous step (step 9. Define focus map), focus on your specimen at the z-position that you want to use for the image acquisition or the top of the desired image-stack for 3D slide scans.

   If you did create a focus map, skip this step.

2. Set slide scan options as desired (see descriptions below).

3. Click Start scanning... to begin the slide scan. The words on the button depend on whether a focus map was created in step 9. Define focus map:

   • Start scanning with focus map

   • Start scanning at current Z (there is no focus map for the scan)

Options

• Keep image open: Displays the final image in the Image Organizer when the scan is complete.

• Remove temporary files: Individual image tiles are saved in a folder until they are compiled into the final image montage at the end of the slide scan. Check Remove temporary files to delete the individual files once the image has been compiled (this will minimize required disk space).

• Preview: Acquires images from each field of view selected for the slide scan, then compiles them into a preview image for review. The preview image opens in the Main window as does the Slide Scan Compile with Preview dialog box.

  Review the image preview and indicate how you want to proceed (click to expand):

  • Filter Setup

    • Background subtraction: Check or uncheck the box to see the results of applying or removing the background subtraction filter in your image preview.

    • Click Setup to change the background subtraction filter parameters and preview the effect of the change.

  • Start Processing: Select options, then click to generate an updated preview or to compile your slide scan:

    • Stitching XY: Stitching uses the image-tile overlap to visually align individual image tiles in the X and Y orientations. It results in better image-tile alignment and cleaner compiled images—we recommend using it in most cases.

    • Compile into single image file: Check the box to compile your slide scan images into one large image.

      Leave the box unchecked and click Start Processing to generate an updated preview with Stitching XY if selected.

  • Postpone Compilation: Image-tile files are acquired and saved, but are not compiled into the full slide-scan image. When you're ready to compile your slide scan into a single image file, use the Scanned slide compiler (go to Image > Compilers) to compile your image.

  • Delete Image: Deletes the preview image.

  • Focus position: [displayed for 3D slide scans only] Click the buttons corresponding to different Z-positions to quickly jump to that location for review of the preview image.

  • Subsampling: View just a portion of the preview image to quickly determine if it meets your needs.

    Zoom in on the portion of the image that you want to evaluate and click Subsample Preview, to quickly load and view just that portion of the image.

    The subsampling feature can significantly speed up the generation of large preview images, especially if filtering has been applied

• Postpone compilation: Image-tile files are acquired and saved, but are not compiled into the full slide-scan image.

  Use the Scanned slide compiler (go to Image > Compilers) to compile your image.

• Compression ratio: Use the slider to adjust the compression and reduce the size of the image files. The current ratio is displayed.

  • In many cases, a compression ratio of 10:1 or 20:1 is appropriate.

• Stitching: Stitching uses the image-tile overlap to visually align individual image tiles in the X and Y orientations. It results in better image-tile alignment and cleaner compiled images—we recommend using it in most cases.

• Flatfield correction: Mitigates uneven illumination artifacts in fluorescence imaging using intelligent, image-data based correction algorithms—we recommend using it for fluorescence imaging

• Capture tissue ___ microns below focus map or top of stack: Type in a number (other than "0") to acquire images at that distance below the Z-plane defined in the focus map or the focal plane (Z position) chosen in step 1 above that establishes the first plane (or top) of image-stack acquisition.

  • Useful for quickly creating the focus map at the top of the tissue, but capturing images at a set distance into the section.

  • If the value is “0”, images are acquired at the designated focus map locations, or at the specified top of stack.

Scan summary

A table showing the slide-scan settings is displayed at the bottom of the workflow window. You may want to review all of the settings before beginning the scan.

We recommend reviewing the estimated disk space required and available. If there will not be sufficient Disk space available, return to step 1. Setup and select file locations that have more than the "Estimated required disk space" available.