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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 I have found that I can get close to the correct focus with a value of - 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.
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The instructions below assume that the sample is stained with a far-red dye like Alexa 647.
- Click View in the Acquisition Control area to get a live
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- feed from the acquisition camera.
- There
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- 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 other than far-red you will need Channel 0.
- The live image won’t show any fluorescence until you illuminate the sample with a laser. Change the percentage power in the Light Control area to 5 to 10%. You can either click on the spin buttons or type in a value and press the Return key. Click on the 640 button to illuminate the specimen.
- 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.
- Turn off the laser so you don’t photo bleach the specimen.
- The best way start acquiring data is to use multidimensional acquisition. Click the Multiple Acquisition Setup button under the focus camera view on the left. In the Acquisition tab, click Single, set imaging mode to Normal, specify the laser power, exposure/frequency and number of images. In Saving, specify the Dataset Tag, which will be the folder where the files are stored, and the Acquisition Tag, which will be the filename. Note that the date will be automatically included in the filename.
You can get more information about multidimensional acquisition in the Knowledge Base on the ONI website.