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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 pattern 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.
- Click the Focus Laser button in the Light Control area on the right.
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.
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.
Note The fluorescence from your specimen will most likely be at a different height to the reflections of the focus laser. You should view the live fluorescence image and use the Z offset field to adjust offset so you fluorescence is in focus. See section on 'Setting acquisition conditions'.
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The following instructions are appropriate for single colour 2D dSTORM using a sample labelled with a far-red dye like Alexa 647.
- 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 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 spinner 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.
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