Assessing Bleach
Assessing Bleach
Quickly Assessing Fluorescence Bleaching in LSM700 Microscopes with ZEN and Fiji
Protocol
Initial Settings
- On the LSM700, make sure you have all settings (Laser, Gain, Pixel Size, Speed) as you would want them
- Do not use averaging or Z stacks
Prepare for acquisition
- Check the Regions tool in ZEN and create a rectangle in a region of your image that contains signal
- In the Regions tool, uncheck Bleach, but keep Acquisition & Analysis
- Check Fit frame size to bounding rectangle of regions
- Check Time Series
- Use 100 timepoints (or more if you want)
Run Acquisition
- Use Start Experiment
- Switch to the Mean ROI tab on the left of the image
Copy the data and run the macro
- On the table, right click and select Copy Data
- Open Fiji and open the macro Assess Bleaching.ijm (Drag & Drop the file into Fiji)
- Run the macro. It will use the data that is currently on the Windows clipboard and return the results
See the results
There will be a message with number of frames before 50% of intensity is lost.
This is also reflected in a new line in a Table called Bleach Assessment
Finally, you can observe the plot to see if the exponential fit worked.
- Last Author
- chiarutt
- Last Edited
- Apr 25 2019, 12:54