Bleaching and ablation procedure

Follow the procedure below to set up photomanipulation experiments like Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Loss In Photobleaching (FLIP) or photoablation.

Make sure the lasers you need for bleaching or ablation are powered on in Zen.
Configure your light path settings for imaging (i.e. lasers, detectors, mirrors, emission filters). You can use Smart Setup, or manually configure them.
If you are going to use the Chameleon multiphoton laser for ablation with infrared light then make sure it is tuned to a suitable wavelength (usually 760 nm).
If you are going to use the Chameleon, then make sure the correct beamsplitting mirror is in place for the invisible laser beam path. For 760 nm this would be the MBS 690+ mirror. If the laser is tuned to a wavelength greater than 760 nm then the MBS 760+ can also be used. The advantage of using the latter is that it is compatible with imaging far red dyes. In contrast, if the MBS 690+ is in place it will block transmission of far red.
Untick the Chameleon laser wavelength in the track unless you want to image using the multiphoton laser.
Set up a Time Series; and a Z-Stack if needed.
Tick the Bleaching box in the multi-dimensional acquisition field. The Regions box will be automatically ticked when you tick Bleaching.
Select an ROI shape from Regions.
Draw at least one ROI on a preview image.
Tick Bleach and, if necessary, Analysis for the region or regions. Analysis is only required when following fluorescence recovery in FRAP and similar experiments.
Set up the bleach parameters in the Bleach Tool according to the instructions below.
Once the bleach parameters, Time Series and Z-Stack settings are configured, click Start Experiment to begin the acquisition.

Bleaching Tool

The Bleaching tool allows you to configure the parameters of the bleach phase of your acquisition. Once you've set the parameters below you can save them for reuse at the top of the tool window.

Start Bleaching after # scans: This allows you to delay the bleaching event so you can acquire a 'pre-bleach' set of data to compare with the post-bleach recovery phase in a FRAP experiment
Repeat Bleach after # scans: This is for use in techniques such as FLIP.
Iterations: Sometimes a single scan of the region is not enough to bleach or ablate, so you can use this feature to repeatedly illuminate the region during the bleach phase.
Different scan speed: This allows you to speed up or slow down the scan rate specifically during the bleach process. It can be advantageous to slow down the scan because this increases the dwell time and hence the amount of energy delivered to the region.
Safe bleach for GaAsP: This protects the Gallium Arsenide Phosphide (GaAsP) detectors from intense reflected light by blocking light from the detectors during the bleach process. This slows the acquisition down so it should only be used if the light levels are so high that the detectors are being saturated and shutting off during the bleach phase.
Different Z Position (µm): Clicking Mark Z will save your current Z position. The focus will then move to that position to carry out the bleach/ablation event.
Trigger in: Allows a remote signal from a port adapter in the Real Time Controller to trigger bleaching.
Trigger out: Can send a signal to the port adapter after a bleach to stimulate another event.
Zoom Bleach: The scanner will zoom into the region to rapidly bleach it. This delivers more energy so iterations may not be necessary. However, the bleaching might bleed into areas outside the region so it can be less accurate.
Excitation of Bleach: Here you can select which wavelengths you want to use to bleach, which ROIs, and whether you want to use different settings for different ROIs.
Test Bleach: This allows you to try out your settings prior to starting the experiment.

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