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The settings in the following guide section 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.
- 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 can should untick Channel 0 in the Image Display Options on the left. For 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 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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