Scan repair

Requires a separate license

Purpose

Use Scan repair to continue a slide scan acquisition that was interrupted prior to completion, or to replace individual tiles that may have been affected by a camera or lighting issue without having to re-run the entire acquisition.

The workflow is designed to guide you through the process of aligning the image with the live camera feed of the tissue on the microscope and starting the repair acquisition.

Procedure

Before you start

If you are using the scan repair workflow to replace specific tile(s) within the slide scan image, you will need to determine the name of the tile image(s) before starting the workflow.

Navigate to the folder where the individual tile images are saved and sort by date. They are located in a folder labeled with a 4 digit number (i.e. 0001, 0002, etc.), within the folder where the slide scan image was saved.
Open each tile image file in Neurolucida to find the affected tile image(s).
Take note of the tile image file name(s) that need to be repaired and delete them from the directory. The scan repair will replace the tile image files with new ones.

Click Scan Repair on the Acquire ribbon to get started.

Scan repair workflow steps

Place reference point

In this step you have the option to place or reset the placement of the reference point.

The reference point needs to be in the exact location it was at the time of the slide scan acquisition.
Click to expand.
- If the repair is being performed in the same session as the acquisition, and the stage and reference point have not been moved, then no action is required.
- If the repair is being performed in a separate session, click in the Main window to place the reference point.
- If there is already a reference point, but it is in the wrong location, click reset reference point then click on the correct location in the Main window.
Align tracing

Use this step if a tracing was created to define slide scan acquisition area that can help with alignment to the live camera feed.
Click to expand.
- Use current tracing to help with alignment: Select to load the data file containing the tracing.
  1. Data file location: If the data file is not currently open, click Browse to navigate to and open the data file.
  2. Click Live Image in the Acquire ribbon to display the live camera feed.
  3. Click Start Joy Free to use the joystick to align the tracing with the tissue being displayed by the live camera feed.
    
    or
    
    Click Align Tracing to use the align tracing tool.
- Do not use tracing to help with the alignment: Select if you do not want to use a tracing for alignment and proceed to the next step.
Load repair data

Click Browse to repair directory and select the folder that contains the individual tile images, and the AssemblyData and RepairData text files. This folder, labeled with a 4 digit number (i.e. 0001, 0002, etc.), is located in the folder where the slide scan image was saved.
Pick image for alignment

Use this step to pick an area on the slide scan image that will help with alignment to the live camera feed.
Click to expand.
- Click Pick image stack for alignment then click on the displayed slide scan image in a spot with visible unique structures that can be used to align the image with the live camera feed.
  
  The image path with be displayed in Selected image stack box.
- Click Pick Different image stack for alignment then click in the Main window to pick a new location to be used for alignment.
Define scan region

This step defines the area around the original slide scan region. This ensures that the microscope will not navigate outside of the defined region for safety of the objective and slide.
Click to expand.
1. Click Define scan region.
2. Use the joystick to navigate, then click in the Main window to designate the upper left corner of the scan region.
3. Use the joystick to navigate, then click in the Main window to designate the lower right corner of the scan region.
4. Click View upper left region or View lower right region to navigate to each corner and confirm the area was defined correctly.
5. Optional: Click Redefine scan region to change the area surrounding the entire slide scan region.
Align image with live

This step is used as the final alignment between the live camera feed and the slide scan image to be repaired.

Accurate alignment of the slide scan image and the live camera feed in the X and Y axes, as well as adjusting the focus position of the live camera feed to match the focus position of the image, is critical in performing a successful slide scan image repair.

Alignment of the live camera feed and the slide scan image is performed using the Blend tool which is automatically enabled.
Click to expand.
- Do alignment of image to live: Select to perform the final alignment before the slide scan repair.
  1. Use slider to adjust blend of the slide scan image and the live camera feed.
  2. Click Start Joyfree to move the stage.
    
    Or
    
    Click Align image to live:
    
    Adjust the blend slider to display the image.
    
    Click on a easily identifiable unique point on the image.
    
    Adjust the blend slider to display the live camera feed.
    
    Click on the corresponding unique point to complete the alignment.
  3. Use the microscope focus to make sure the live camera feed is in the same focus position as the image. To do this, pick one object (such as a cell) that is in focus in the image and adjust the focus on the microscope until that object is in the same focus plane in the live camera feed.
- Skip alignment of image to live: Select this option if the image and live camera feed are already in alignment. This is usually the case if the repair is being performed in the same session as the slide scan acquisition and the stage and reference point have not been moved.
Auto-align image with live

This step is used to automatically align the image and the live camera feed in the Z axis. This ensures that the focus of the repair tile images will align with the focus of the original slide scan image.
Click to expand.
- Do auto-alignment: Select to acquire an image stack for Neurolucida to use to automatically align focus of the live camera feed with the slide scan image.
  1. Click Live image: to display the live camera feed.
  2. Focus to top of stack and click Set top of stack.
  3. Focus to bottom of stack and click Set bottom of stack.
  4. Channels for alignment: Select the channel to use for the focus alignment from the dropdown menu.
  5. Subset of images to acquire: Select a subset of images to be acquired for the alignment stack from dropdown menu. For example, choose 3 to acquire every third image. This can save time if the original slide scan image consists of a lot of image planes.
  6. Click Acquire stack and align.
- Skip auto-alignment: Select this option if the focus alignment is accurate and the automatic alignment is not necessary.
Start the repair
1. Adjust the microscope focus to just below the bottom Z position of the of the slide scan image.
2. Click Set safe lower limit to ensure the microscope will not navigate below this set Z position for the safety of the objective and slide.
3. Click start the repair.