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- Click the View button in the Acquisition Control area in the lower right of the UI to begin a live feed from the acquisition camera. You might need to scroll downwards in order to see the View and Acquire controls.
- There will be two live images, which correspond to two simultaneous colour channels. In the top left next to the live images there is a small image with green and red rectangles. The two channels are split using a dichroic mirror and projected onto different regions of a single camera. The rectangles show the relative positions of the two channels on the camera chip.
- For dSTORM using a far-red dye you only need Channel 1 (the one outlined in red), so you should untick Channel 0 in the Image Display Options on the left. This will prevent you from acquiring all the images from an unused channel. If you are using dyes emitting wavelengths shorter than far-red then you will need Channel 0.
- 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. It is best to use a lower power like 10% to avoid photobleaching while setting up your specimen. The A and B buttons allow you to create two laser illumination conditions, so you could have A set to 100% and B set to 10% and use the former for imaging and the latter for setting up. Click on the button above the laser (e.g. in this case the red 635 button) to switch on the illumination. The microscope will emit a high-pitched noise while the laser is operating. This is normal.
- 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 from 0° to 53°.
- Your sample will probably not be in focus initially. Normally the focus can be adjusted by changing the value in the Z: field (see area outlined in red in the Position Control image below), but if Z lock is active the focus cannot be changed this way. Instead you should adjust the Z offset, which offsets the focus position from the fixed Z Lock position.. 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 by clicking its button in the light control area, so you don’t photobleach the specimen.
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