= 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 {nav Regions} tool in ZEN and create a rectangle in a region of your image that contains signal - In the {nav Regions} tool, uncheck **Bleach**, but keep **Acquisition** & **Analysis** - Check **Fit frame size to bounding rectangle of regions** - Check {nav Time Series} - Use 100 timepoints (or more if you want) === Run Acquisition === - Use {nav Start Experiment } - Switch to the **Mean ROI** tab on the left of the image === Copy the data and run the macro === - On the table, {nav right click} and select **Copy Data** - Open Fiji and open the macro `Assess Bleaching.ijm` (Drag & Drop the file into Fiji) {F10627035} - Run the macro. It will use the data that is currently on the Windows clipboard and return the results === See the results === {F10627023, size=full} 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.

Document migrated to https://wiki-biop.epfl.ch/en/Teaching/PCB/assess-bleach