Loading images from an external image source with Optical Fractionator

can load an image stack from an external image source at each site before starting counting if the Open Confocal module is enabled.

1. Start the Optical Fractionator workflow.
2. Run through the workflow steps using the camera and camera lenses to trace the region of interest and continue to the Define the Counting Frame Size step.

The lens specified in the Set Microscope to High Magnification step is a lens for the camera, not for the external source.

3. At the Define the Counting Frame Size step, you have the option to move the counting frame.
   • Since you will be counting using images that may get loaded slightly away (offset) from the upper left corner of the tracing window, place the counting frame so that it overlaps images acquired externally.
   • If you don't know the offset between the images and the upper left corner of the window, determine it now.
   Determining the offset
   1. Acquire an image via the external source and use the objective associated with the High Magnification lens.
   2. Select File>Open>Image. The Image Scaling and Placement dialog box is displayed.
   3. Select Recalculate the placement of the image... / Override the X,Y Scaling / the lens for the external image source and that objective.
   4. You should now be able to see where the images are going to get loaded relative to the upper left corner of the tracing window. Move the counting frame so that it overlaps this image.
4. Continue the workflow to the Count Objects step.
5. Before you start counting, click Show Advanced Features, then check Load acquired stack at each site.
   • Load External Stacks is enabled when you select a contour or a previous probe run.
   • To select image file(s) to open for each site, check Scan directory for new image file(s).
     • If you selected multiple files to open into an image stack at the first site, you see this dialog:
       1. This dialog is an indication that the stage is now at a new site. Acquire the images for the site then click OK.
       2. The Order of Files for Stacks dialog box is displayed.
     • If you selected one image file (a multi-image file) to open at the first site, you see this dialog:
       1. The dialog disappears once the new image is in the directory.
       2. The Image Stack Open dialog box is displayed with the image file pre-selected. Click OK.
6. Click the Start Counting button. As you approach each site, the program verifies that there is a stack loaded for the site. If not, you're prompted to load a stack.
Loading a stack
1. Use the external image source to acquire your stack, save the stack, then return to .
2. Select the stack to load.
   • With the first stack you load, the Image scaling and placement dialog box appears. Select Recalculate the placement of the images... / Override the X,Y Scaling / Specified lens / the lens for the external image source and the associated objective.
   • After the first site, the program uses the settings defined for the first stack.
3. If you make a mistake, click Load External Stack or Remove Stack .