Assessing Bleach

Updated 2,030 Days AgoPublic

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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) ready
• Do not use averaging or Z stacks

Prepare for acquisition

• Using the "Regions" tool in ZEN, create a rectangle in a region of your image with signal
• In the Regions menu, uncheck "Bleach", but keep "Acquisition" & "Analysis"
• Check "Use bounding rectangle blablabla"
• Check "Time Series"
• Use 100 timepoints (or more if you want)

TODO, illustrate image better

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

See the results

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