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Rightclick in the SW and in the acquisition control open A1plus Stimulation and ND Stimulation panel
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Dock it somewhere in the SW
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Optimize the laser pw and scanning parameters as usual.
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Capture image with those settings
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press Ctrl+Shift+R to display ROI Editor to draw 3 ROI
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Draw a ROI of 1-3 um in diameter and then right-click and duplicate twice, so you have in the end three ROI
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Should be small enough ~1-3 um. If too large, the diffusion won't be correct because your molecule will diffuse into the ROI, while bleaching will be still running.
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Position in the area, where you want to bleach signal.
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Rightclick on the ROI > Assign to: A1 > S1/S2/S3
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Parameters of the Stimulation/Bleaching settings you can find in this window
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Choose the wavelength for bleaching
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Change the laser power - Aim for ~50–70% intensity loss. This typically requires several test runs and intensity measurements.
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One of the ROI place where is only background signal
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Right-click → Assign to Background.
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Last ROI will be placed in the region, where is your specific signal, but no bleaching will happen
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Right-click → Assign to Reference.
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The reference ROI should show minimal intensity drop during the experiment. If the reference curve shows bleaching, adjust imaging parameters
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The acquisition consist of three phases: Pre-bleaching, Bleaching, Post-Bleaching (Recovery)
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You can rename phase depending on whether you perform stimulation or bleaching.
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Define the timelapse duration before bleaching and the interval between frames.
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5-10 loops to measure the intensity before bleaching
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Define the timelapse duration after bleaching, where signal recovery will be captured. Use the same interval as in Phase #1.
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In the bleaching phase, you can use a very short duration (~100ms). SW redefines the duration, so the bleaching occurs only once
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Check the relevant boxes to enable Time Measurement.
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Click Apply Stimulation Settings, then proceed with Run.
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After the experiment, the Time Measurement window opens automatically, showing intensity curves for all ROIs.
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You can export the data in the excel sheet here.
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Reference ROI: Should have minimal drop of the intensity, otherwise you bleach already with your acquisition settings: If yes, you have to adjust them to avoid the bleaching or perform Reference correction during Data analysis
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Background ROI: could be used to subtract all non-specific fluorescence and camera/detector noise and backgroud signal coming from the sample, so that only the true signal from your sample is analyzed.
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