9. Define focus map

Overview

When this workflow step is active, the default display will show boxes corresponding to all of the scan sites (or fields of view) that will be included in the slide scan.

In this step, you indicate a uniform Z position in different areas of the specimen by selecting focus sites and bringing them into focus to create a "focus map" for the slide scan. The focus map can be used as the upper starting position (Z position) for image acquisition or you can set the slide scan to start acquiring images at a set distance in microns below the Z position defined in the focus map in the next workflow step (see Capture tissue __ microns below...). Note that:

It's important to set the focus site at the same relative Z-position at all sites to obtain a final image that accurately reflects the specimen.
If your histological specimen is flat, you can likely use fewer focus sites, however, if it is wavy or uneven, you will need more focus sites.
It may save you time to set the focus map at the top of your specimen. If you want to acquire images at a set distance below the top of the specimen, you can type in a value for the setting, Capture tissue ___ microns below focus map or top of stack in step 11. Start scanning.

Procedure

Focus sites

Choose your focus sites using one of the following methods:

Note: Focus sites will be displayed onscreen with a heavier outline. If they are difficult to distinguish from scan sites, click the box to Show only selected sites in the Display options.
- Click scan sites with your mouse to manually select them as focus sites.
- Use markers as focus sites: Click to define focus sites in scan sites that contain markers placed during workflow step 3. Trace .
- Randomly select focus sites: Click 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".
    - Note that 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".
    - Note that you must enter fewer sites than the value indicated at the bottom of the dialog box.
- 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

Set the focus at each focus site.
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).

Other options

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.

Focus Adjustments

Manually, every: Check the box to add manual focus adjustments to your scan and enter the number of images between each manual focus site into the box.

When enabled, the acquisition stops at the specified increments and you are prompted to bring the tissue into focus in order to continue the slide scan.

Manual focus adjustments are helpful if your microscope system is prone to stage drift.
You may also add manual focus adjustments to simply include some focus checkpoints in your scan.