Configuration & Calibration

scanfrap_configuration

The scanFRAP section (A) in the Configuration tab allows you to adjust the point density (space between points) for photomanipulation. The X and Y spacing refers to the density of filled forms. For lines or unfilled forms, the line/Y spacing is considered.

Fire Preview can be activated to test the scanFRAP settings. When activated, the laser will emit light on selected ROI.

Note: The Fire Preview option requires (i) some ROI to be drawn on the image and (ii) the correct channel to be selected.
Before starting photomanipulation experiments, the scanFRAP must be calibrated. This semi-automated step is necessary in order to achieve a high level of accuracy on galvanometric mirrors.
  1. Manual calibration
    After clicking on the FRAP in the calibration list (B), you have to:
    1. Select the camera in the drop-down list.
    2. Select the pre-saved channel to use
    3. Select a previously done calibration or make a new one
    4. Click on Confirm.
    scanfrap_manual_calibration
    Fastpath: It is recommended to perform this calibration using a fluorescent slide to obtain a strong signal and avoid laser phototoxicity.
    Important: It is recommended to recalibrate each time the scanFRAP is used to optimise the accuracy of the system.
  2. Automatic calibration
    Notice: It is important to ensure that the focus is good throughout the calibration process. The laser is most accurate when in focus. Do not hesitate to adjust the focus before the calibration to get the best result.
    1. Click on Live if it is not already done (then you see Stop).
    2. Use the virtual joystick to move the laser pointer to any of the virtual crosses on the screen.
    3. When the laser pointer is on the cross, click Select. Repeat steps a and b three times.
    4. A fully automated protocol will make a pattern on the image. You have to validate the regularity of this pattern and click on Confirm.
      Remember: If you find that the calibration is not good enough you can reprocess the calibration with the detection algo tab.
    scanfrap_automatic_calibration
Tip: To validate the calibration, do not hesitate to do some tests. For this, place some ROI on the live image and click on Fire Preview.