Phriction Projects Wikis Bioimaging And Optics Platform Teaching Assessing Bleach History Version 3 vs 4
Version 3 vs 4
Version 3 vs 4
Content Changes
Content Changes
= 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)
{F10627033} 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)
{F10627035}
- Run the macro. It will use the data that is currently on the Windows clipboard
=== See the results ===
{F10627023, size=full}
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
= 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 resions**
- 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.
= 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) readyas you would want them
- Do not use averaging or Z stacks
=== Prepare for acquisition ===
- Using- Check the "{nav Regions"} tool in ZEN, and create a rectangle in a region of your image withthat contains signal
- In the {nav Regions menu} tool, uncheck "**Bleach"**, but keep "**Acquisition" & "** & **Analysis"**
- Check "Use**Fit frame size to bounding rectangle blablabla"of resions**
- Check "{nav Time Series"}
- Use 100 timepoints (or more if you want)
{F10627033} TODO, illustrate image better
=== 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}
MThere 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.
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