Nikon AXR FRAP procedure

This page is a brief introduction to doing FRAP experiments with the Nikon AXR or Nikon AXR NSPARC microscopes.

Setting up FRAP in Nikon Elements

• Open the AX stimulation dialog. This can be found in the View menu on the top bar of Nikon elements. Another way to access it is to right click on the area where the image shows up but not on the image directly.

• Open the ND stimulation dialog. This can be found in the View menu on the top bar of Nikon elements. Another way to access it is to right click on the area where the image shows up but not on the image directly.

• Now you should have two dialog boxes as below:

• The next step is to create an ROI on your image where you will do the FRAP stimulation and assign it to the current configuration as follows:

• Last you need to be in Galvano mode to perform FRAP

Basic FRAP experiments

To set a basic FRAP experiment and target a small ROI in your sample:

• Find an area in your sample that you don't care too much about to test the FRAP settings on. 
• Open the AX stimulation and ND stimulation dialog boxes.
• Define your ROI.
• Set the Dwell time to 1us in the AX stimulation dialog box. You can also set the laser power to 100% or middle of the range. This is your starting point.
• Go in the ND stimulation dialog box and set up and Acquisition then a Stimulation then an Acquisition again as shown in the screenshot above. The first acquisition is the image of your sample before you perform FRAP. The stimulation performs FRAP and the last acquisition is an image of your sample after performing FRAP.
• In order to increase the intensity of the photobleaching occurring during the FRAP stimulation you can:
  • Increase the number of loops in the ND stimulation dialog box. 
  • Increase the laser power.
  • Increase the dwell time. This will significantly increase the time it takes to perform FRAP. And the values you can enter for the dwell time are discreet, they are the same as the dwell time values under the Galvano setting. Entering any other value will not work, it will get back to one of the preset values as below.