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1. The Nanoimager has no eyepieces so focusing must be done using a camera image. This can be done using sample fluorescence, but I’ve found that it is a lot easier to use the focus laser, which reflects at the glass/water interface of the specimen and forms an image of a spot on the focus camera. The Focus Camera view is in the top left of the NimOS user interface. The focus camera view will just display a live repeating noise image until the focus laser is switched on.
   

   Info
   If the focus camera view is not visible, select Show Focus Camera View from the Instrument menu.

2. Click the Focus Laser button in the Light Control area on the right.
   

3. You will probably initially see a faint diffuse signal at the top of the focus camera view. This is what the camera view looks like when out of focus. Type -300 µm into the Z field and gradually increase the value using the spinner (making it more positive) until the laser spot comes into focus. The correct position is when the laser spot is at its smallest and brightest (see 'in focus' image below).
   

   Info
   The 'in focus' position will be different for different imaging vessels. I have found that I can get close to the correct focus with a value of about - 250 to -300 for ibidi µ-slides and +16 for the bead channel alignment slide. The latter is larger because the coverslip is mounted on spacers so the stage has to be moved to a higher position.

4. You can prevent the focus drifting by locking it to the laser reflection position. Click the Set Focus Ref. button in Position Control. The Nanoimager will auto focus on the focus laser reflection. When the auto focus is complete, click the Z Lock button to prevent further drift.
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   Note
   The fluorescence from your specimen will most likely be at a different height to the reflections of the focus laser. The Z offset field can be used to adjust the height so sample fluorescence is in focus. See section on 'Setting acquisition conditions'.

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Setting acquisition conditions (for single colour 2D dSTORM)

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1. 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.
   
2. 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.
   
3. 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.
   
4. 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. 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.
   
5. 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°.
   
6. 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 as explained in the section on focusing, 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.
   
7. 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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