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- Click View in the Acquisition Control area to begin 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 emitting wavelengths lower than far-red you will need Channel 0.
- The live camera image will only show noise until you illuminate the sample with a laser. Change the percentage power in the Light Control area by either clicking on the spin buttons or typing in a value and pressing the Return key. The laser power in the image below is set to 100% for dSTORM acquisition, but it is best to use a lower power like 10% to avoid photobleaching while setting up your specimen. Click on the button above the laser (e.g. red 640 button) to switch on the illumination.
- 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.
- Once you're in focus turn off the laser so you don’t photobleach the specimen.
Acquiring images
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Acquisition Control
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Acquisition Control is the most obvious way to start an acquisition because it is easily visible in the user interface. However, the best way I've found to acquire images is to use Multiple Acquisition Setup, which is explained later. |
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The laser doesn't automatically come one when you press Acquire, which is why you need to turn the laser on in the Light control area. Multiple Acquisition Setup handles this better.
Multiple Acquisition Setup
The settings in the following guide are appropriate for single-channel 2D dSTORM of a sample labelled with a far red dye like Alexa 647. Your sample or procedure may require other settings, such as an additional imaging channel, different laser wavelengths and powers, or 3D acquisition. If you need to use different settings, then you can either look for more information on the wiki or in ONI's knowledgebase (login required) or contact the light microscopy email: lmcb-lm-help@ucl.ac.uk.
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