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- If necessary, boot up the laptop computer and login to the ONI account.
- Double-click the NimOS icon on the desktop to launch the acquisition and analysis software.
- Click the Acquire button in the top left and then click Connect to Microscope.
- NimOS will open the dialogue box below asking you to check the stage before it connects to the device to prevent damage.
- Push the safety interlock slider to the in position and lift the lid of the chamber to make sure the area around the stage is clear and that all samples have been removed. There should be two stacks of small disc magnets, one stack of three on either side of the stage. They are there to secure the specimen and should be left in place.
- Leave the lid open and click Confirm and Connect on the computer screen. There will be noise as the Light Engine starts up and messages will appear in NimOS as the computer connects to the different components.
- You will see that the stage moves to very high position as part of its start-up. This is why the stage needs to be clear prior to initialisation.
- In the Temperature Control area on the left of the user interface, set the Target Temperature to 31ºC or 37ºC and press the Enable Control button (the button with change colour and say Disable Control once pressed). The Nanoimager operates at a temperature around 6 degrees warmer than ambient and the temperature control must be set slightly above that to maintain stability. This is something to take into consideration when planning applications where temperature is important.
Once the temperature has stabilised the control can be disabled.
Note ONI say that it will take the microscope 60 to 90 minutes for the microscope to reach 37°C and stabilise.
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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. 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.
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