You can get more information about the procedures described on this page in the Knowledge Base on the ONI website, although you will need to login to see the information. Sign up with ONI for an account.

Initial start-up procedure

1. The Nanoimager is normally left on with the PC logged in, in which case you can go directly to step 5.
2. If you do need to turn on the Nanoimager, first make sure the power switch on the back of the Light Engine is switched on.
   
3. Boot up the PC by pressing the power button
4. Login to the ONI account.
5. Double-click the NimOS icon on the desktop to launch the acquisition and analysis software.
   
6. Click the Acquire button in the top left of the user interface (UI) and then click Connect to Microscope.
   
7. Click on 'OK' when the "could not identify instrument" window pops up                                               
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8. Select 3D_QZX2P3SM for 3D single molecule imaging and QZXZP3SM for any other type of imaging (including 2D single molecule imaging)
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9. Click "Connect to Microscope"
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10. NimOS will open the dialogue box below, asking you to check the stage before it connects to the device to prevent damage.
    
11. On the Nanoimager microscope, push the safety interlock slider to the 'in' position. This unlocks the lid and prevents the imaging lasers from coming on.
12. Lift the lid of the chamber and 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 will be used to secure the specimen later and should be left in where they are.
    
13. 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.
14. You will see that the stage moves to very high position as part of its initialisation. If there was a sample on the stage and the lid had been closed there would have been a significant risk of the sample hitting the inside of the lid, possibly damaging the stage. This is why the stage needs to be clear of specimens prior to initialisation. The stage can also be initialised with the lid closed provided there is no sample on the stage.
15. 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 will 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.
    

16. 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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1. 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.

2. Click the Focus Laser button in the Light Control area on the right.
   

3. 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.

4. 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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1. Click the Multiple Acquisition Setup button under the focus camera view on the left.
   
2. In the Acquisition tab, click Single, set imaging mode to Normal, adjust the 635 laser power to 100% and make sure the button above the percentage slider is clicked to activate the laser (text should be white, colour should be bright). Set the exposure and number of images.
   
3. Ignore the Priority and AutoFocus and Z Reference tabs. They are not important for conventional 2D dSTORM.

4. In the Saving tab, specify the Dataset Tag and the Acquisition Tag (see Acquisition Control above for an explanation of the tags). If you are logged into your User login all files will be automatically saved in your folder.
   

   Note
   By default NimOS performs molecular localisation on data as it is acquired. If you don't want it to do that, tick the box that says Disable real-time localization.

5. Click the Start button at the bottom to begin the acquisition.
   

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6. Once Multiple Acquisition Setup has run the lasers in Light Control will be greyed-out. You need to click the button that says 'Enable active lasers' before imaging again.
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