FRAPPA setup


Resulting difference image stack of automated calibration routine.

Uniform fluorescent slide

  1. Focus on the Ethidium Bromide calibration slide with the eyepieces using transmitted light. The liquid layer is thin and can require refocusing every few minutes.
  2. Use a camera exposure of 200-500ms for imaging. As with most image processing calibration algorithms, a fair amount of signal is necessary for an accurate image registration.

First time hardware setup


These steps are only necessary when mechanically aligning the FRAPPA galvo system with it detached from the microscope.

  1. Select the “Illumination setting (during pulse)” and “Illumination setting (during imaging)” in the Configuration tab.
  2. Adjust the “Rest Position” angle in the Setup tab to center illumination on the C-mount target on the FRAPPA excitation aperture.
  1. Click the “Save” button when you are happy with the bypass position.
  2. In the Configuration tab, set the “Dwell time [us]” to 10,000 (without the comma) for an EMCCD, or 100,000 for other cameras.
  3. Click “Pulse” back in the Setup tab and adjust the offsets (by 0.1 increments) to center the laser spot on the center of the image.
  4. Cycle MetaMorph so that these setup values are written into the mmproc.cfg file, just in case the software crashes. The more convenient way of using File > (holding Shift key) + Exit unfortunately does not save FRAPPA settings.

Steps to calibrate


On multi-user MetaMorph systems, it is recommended to create a special user to use “Targeted Illumination” hardware, like the FRAPPA, Mosaic and Micropoint.

The “Targeted Illumination” calibration settings are stored in each user’s mmproc.cfg, and there are no “Backup...” or “Restore...” buttons (or journal functions) to make it possible to to keep calibrations up-to-date for every user.

  1. A safe value for “Range for calibration point” for EMCCDs is 20% to 30%
  2. Enable “Display images during calibration”. This allows you to see the difference images as the software is doing the automated calibration, providing useful diagnostic information.
  3. There should never be any need to use “Manually click on calibration points” with the FRAPPA since we have good control over the laser settings.
  4. Enable autoscaling in the Acquire window’s Display tab.
  1. Select the “Mag:” setting for the current objective + camera combination in use.


MetaMorph ties together the name of the Magnification setting to the Calibration setting; i.e. the Calibration name is the same as the Magnification setting. When using multiple cameras with the Targeted Illumination device (such as a multi-camera Confocal or back-port FRAPPA system), create a Magnification setting for each camera like “100x EMCCD” and “100x sCMOS”

  1. Finally, click “Create Setting...”. During calibration you will see each point pulsed in a 3x3 grid.
  1. The new calibration setting appears in the “Coordinate system setting” dropdown list, if successful.
  2. Repeat steps 5 and 6 for all objectives with sufficient NA.
  3. Important: Change the “Dwell time [us]” back to 100, otherwise trying to FRAP 2D regions will take several minutes instead of the low order of seconds!
  4. Optionally, you can draw a small region of interest and click “Pulse” to test the calibration. There are 2 different “Pulse” buttons: one under the Configuration tab and one under the Location tab. Use the “Pulse” button in the Location tab.