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The instructions below assume that the sample is stained with a far-red dye like Alexa 647.
- Click View in the Acquisition Control area to get a live feed from the acquisition camera.
- There will be two live images, which correspond to two simultaneous colour channels. For dSTORM using a far-red dye you only need Channel 1, so untick Channel 0 in the Image Display Options on the left. For dyes other than far-red you will need Channel 0.
- The live image won’t show any fluorescence until you illuminate the sample with a laser. Change the percentage power in the Light Control area to 5 to 10%. You can either click on the spin buttons or type in a value and press the Return key. Click on the 640 button to illuminate the specimen.
- Focus on your specimen. If you are using Z lock you may need to adjust the Z offset as explained in the section on focusing.
- Turn off the laser so you don’t photo bleach the specimen.
- The best way start acquiring data is to use multidimensional acquisition. Click the Multiple Acquisition Setup button under the focus camera view on the left. In the Acquisition tab, click Single, set imaging mode to Normal, specify the laser power, exposure/frequency and number of images. In Saving, specify the Dataset Tag, which will be the folder where the files are stored, and the Acquisition Tag, which will be the filename. Note that the date will be automatically included in the filename.
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