OPEN
CLOSED
Nikon TIRF * Protocol for FRAP
This is the simpler of two protocols. The more complicated one is here.
Step-by-step
Put the bilayer on the microscope and set up imaging with the AOTF as low as you can set it to get images with an average intensity value of at least 500. For instance, AOTF 1, camera sensitivity EM 3MHz, Gain 170, Conversion gain 2.4x, exposure time 100 ms.
To see the bilayer, you may need to adjust the LUT dramatically.
Remember to right click on the the button at the top of the screen to save the microscope and camera settings!
In the "ND Acquisition" tab set the Lambda to only image the one channel.
In the "ND Acquisition" tab set the Time for two time lapse sequences.
The first sequence should have a 2 seconds interval with one loop that takes two images.
The second sequence should have intervals of 10 to 30 seconds for a few minutes depending on how temporally precise you want to record the recovery.
Use the camera button to take a single snapshot which is the prebleach image.
To set the bleaching, set the laser in the AOTF window to 100%.
Set the TIRF angle in the Ti window to be a little out of TIRF. (For example, if 1755 is TIRF, set to around 1900.)
Pop to the top the ND acquisition window so the "Run Now" button is visible for quick access.
Push the field diaphrapm slider all the way in to close it.
|
|
OPEN |
CLOSED |
Click the "Aux1" button to bleach the spot. (Or whatever the button is labeled that opens the laser without enabling the camera-- DO NOT BLAST THE CAMERA WITH BRIGHT LIGHT LIKELY WITH LASER AT 100%!)
Time the bleach (i.e. count to 4 slowly).
To stop bleaching and take the first image, click "Run Now" in lower right of "ND Sequence Acquisition" window.
Make sure the first image has been collected. As soon as you see it on the screen, pull open the field diaphram slider. You should be able to do this before the second of the two second time lapse images is recorded. (If you need more time, change the delay of the first time lapse sequence to 3 or 4 seconds.)
Sit back and let the microscope collect images.
If you want to save the FRAP images, make sure you save both the prebleach snapshot and the recovery time lapse.
If there is recovery, continue with your experiment. If there is not recovery, try another bilayer.
content last updated 20111116
comments, questions, suggestions: Michael.Cammer@med.nyu.edu