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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 on the left of the NimOS GUI. You will just see camera noise unless 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. Change the Z position in the Position Control area on the right-hand side of the screen until there is an image of a small laser spot in the focus camera view. Type -300 into the Z field and gradually increase the value (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.
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 that the fluorescence from your specimen will most likely be at a different height to the reflection of the focus laser. You should use the Z offset field to adjust the focus offset so your fluorescence is in focus.
Single colour dSTORM
Setting up the acquisition
The instructions below assume that the sample is stained with a far-red dye like Alexa 647.
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