Setting up to obtain images from an external image acquisition source

Purpose

In this scenario, our software controls the stage of your external image source (e.g., confocal microscope with no camera).

Procedure

To define your external image source as the sole source of images, follow the instructions below.

• If your system has no camera, ignore the steps for camera setup.
• Define the size of the image to center on the optical center via External Image Source Setup.

Detailed setup instructions

1. Configure the stage (go to File > Hardware setup > Stage).

   Configure for the Virtual Z stage if your external image source has a built-in Z (focus) stage.

2. Configure the camera.

   • The camera image must be oriented the same way as the confocal image.

   • Use the camera option to flip the image if necessary.

   • The image size of the camera does NOT have to be the same as the image size of the external source. The camera and external image source do need to be lined up with the movement of the stage.

3. Go to File > Hardware setup > External image source to specify the size of the external images. Check the box Loading images from an external source while controlling the stage.

4. Create lenses for the camera.

5. Create lenses for the external image source.

   You can also define lenses with acquired images; the acquired image must be displayed at 1:1.

6. Perform a parcentric calibration on all lenses as follows:

1. Make sure parcentric calibration is off.
2. Start a new data file closing all images.
3. Place a slide on the stage (maybe a grid slide) and position it in such a way that a given point can be found at all magnifications.
4. Make sure you have specified the correct image size (cf. Step 2).
5. Turn on Synchronize stage and images (Type Synchronize stage in the Search bar to access this command).
6. Do not move your stage in X or Y and do not move any images until calibration is complete.
7. Select Acquire>Capture>Use external.
8. For each objective:
   1. Move to the objective.
   2. Set the lens for that objective and the external image source.
   3. Acquire an image using the external image source and load the image in the program using the current lens for scaling.

      If your external image source and your camera have the same size image, select Place Image in upper left hand corner of the main window in the Image Scaling and Placement dialog box.

9. Now perform the parcentric calibration for all the lenses.
   • When working on a lens for the external image source, use Image Organizer to display only the acquired image from the external image source for that objective.
   • When working on a lens for the camera, display a live image.
10. When calibration is completed, turn OFF Synchronize Stage and Images.

7. Test your setup as follows:

   1. Select an objective to test.

   2. Turn live image on and bring an object or feature into focus.

   3. Acquire an image from your external image source and save it to an image file.

   4. Select Acquire>Capture>Prepare external.

   5. Select the lens for that objective and the external image source.

   6. Go to File > Open > Image and open the image file you just saved; use the scaling from the current lens.

      If the Image Scaling and Placement dialog box appears, select Place Image in upper left hand corner of the main window.

   7. Turn live back on and select the lens for the camera.

   8. Verify that the image you just loaded matches the live image using Acquire > Display > Blend.

   9. Select File > Close All Images.

   10. Verify that live image is on.

   11. Go to File > Open > Image and open the image you just closed. The Image Scaling and Placement dialog box opens.

       • Select Recalculate the placement of the image assuming the image should be centered on the optical center.

       • Check the box to Override the X and Y Scaling.

       • Select Specified Lens.

       • Select the lens defined for that objective and using the external image source.

       • If you succeeded in performing the parcentric calibration, the correction values should appear in Additional Offsets.

   12. Verify that the image matches the live image using Acquire > Display > Blend.