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- Click the View button 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 you can untick Channel 0 in the Image Display Options on the left. For dyes emitting wavelengths shorter than far-red you will need Channel 0 to be ticked.
- The live camera image will only show noise until you illuminate the sample with a laser. Change the percentage laser power in the Light Control area by either clicking on the spinner or typing in a value and pressing the Return key. The 640 nm 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. in this case the red 640 button) to switch on the illumination.
- Generally speaking for single molecule techniques it is better to increase the contrast by operating the microscope in TIRF mode. In the Optical Control area on the right of the user interface, change the Target Illumination Angle to 53°.
- Unless Z Lock is active you should focus on your specimen by changing the value in the Z: field (see area outlined in red in the Position Control image below). If Z lock is active the Z: position cannot be changed, so you should adjust the Z offset as explained in the section on focusing. Both the Z field and Z offset are highlighted with a red outline in the Position Control image below.
- Once you're in focus turn off the laser so you don’t photobleach the specimen.
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