Assessing Bleach

Updated 1,894 Days AgoPublic

Actions

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 resions
• 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.