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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 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.
- 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 µibidi µ-slides and +16 -27 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.
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