Slide scanning workflow >

8. Sample acquire

Overview

Acquiring a large image can be time consuming; we recommend that you conduct a sample acquire to test the acquisition settings before performing the full slide scan.

This can be helpful to:

• Evaluate whether scan settings, for example overlap/trim and stage-delay settings are appropriate.

• Review whether background correction is needed and/or evaluate the result of your background correction settings (5. Scan settings).

• Preview the effects of Stitching XY and Background subtraction.

Procedure

Check the box to Acquire sample image.

To skip this step, do not check the box and click Next Step to continue the workflow.

Acquire

1. As per the onscreen instructions, move the stage to bring a portion of the specimen into view.

2. Choose a scan size using the drop-down menu. 2 x 2 grid is often a sufficient area for the sample.

3. (optional) Set up focus map...: Click to set up a focus map for the sample acquire. Additional options will be displayed in the window as follows:

   Click to view focus-map setup instructions

   Focus sites

   • Right-click scan sites with your mouse and choose Add to Focus Site List to manually select them as focus sites.

   • Right-click in the display window and choose Randomly select focus sites to have Neurolucida choose focus sites randomly; the Random Focus Point dialog box opens.

     Total sites: The total number of scan sites is shown near the top of the dialog box.

     Click a radio button to indicate how you want the software to randomly choose focus sites:

     • by percent: Enter a percentage of the scan sites that you want to randomly select as focus sites. An approximate number of sites is displayed, "About N sites". You must choose a lower percentage than the value indicated at the bottom of the dialog box.

     • by amount: Enter the number of scan sites that you want to randomly select as focus sites. An approximate number of sites is displayed, "About N sites". You must enter fewer sites than the value indicated at the bottom of the dialog box.

   • Use markers as focus sites: Click to define focus sites in scan sites that contain markers placed during workflow step 3. Trace .

   • Clear all focus sites: Click to start the focus-site selection process again if you're not satisfied with the focus map created.

     There are more options for setting and reviewing focus sites when you right-click; using right-click options may help you speed up your work.

   Perform focus

   1. Focus/Refocus at selected sites: Click to bring the desired focal plane into focus at each focus site; the Focus Site dialog box opens and the stage moves to bring the first focus site into view.

   2. For 3D scans, adjust the focus to establish the top plane of the image stack and bring it into clear view.

      For 2D scans, adjust the focus to clearly see the desired focal plane.

   3. Click Next to move to the next focus site and adjust the focus as in step b.

      Other buttons in the Focus Site dialog box:

      • Previous: Revisit the previous site

      • Skip: Skip the current site ; that focus site will be removed from the focus map.

      • Cancel: exit the focus map; your data will be lost.

      • More: Open Device Control options, including Multichannel Control, Camera Settings. You can also run a previously set up Device Command Sequence or Change the State of a Device.

   4. Set the focus for each focus site. When all the focus sites have been set, the focus grid is displayed with Z-values at each scan site.

      Focus sites display Z-values in yellow; all other sites display interpolated Z-values (their color depends on whether the Show heat map option is enabled).

   Display

   Show only selected sites: When checked, only the scan sites selected as focus sites are displayed. This can be useful for very large scans and/or when you are only interested in viewing the focus sites.

   Show heat map: When checked, Z-values are displayed as an interpolated heat map.

   • Type in a Heat map threshold value and click Update to view the heat map based on the new threshold value:

   • Yellow represents the focus sites

   • Green Z-values differ from adjacent sites by less than the threshold value.

   • Red Z-values differ from at least one adjacent site by the threshold value or more.

4. Start Scan: Click to start the sample scan. A progress window will be displayed while images are captured.

5. When scanning is complete, the sample image appears in the Main window and the SlideScan Compile with Preview dialog box opens.

   Review the sample-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 sample 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 sample slide-scan image tiles.

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

   • Postpone Compilation: Sample image-tile files are acquired and saved, but are not compiled into a slide-scan image. Note that clicking this button will close the dialog box.

     If desired, you can compile your sample slide scan into using the Scanned slide compiler (go to Image > Compilers).

   • Delete Image: Deletes the sample 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.

   • If needed, modify slide scan settings (e.g., in step 5. Scan settings and step ) and repeat the Sample Acquire in this workflow step to optimize the slide scan.

   See also Slide scanning: Troubleshooting for fluorescence microscopy

   or Slide scanning: Troubleshooting for brightfield microscopy for troubleshooting suggestions.