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                                               
   
8. Select 3D_QZX2P3SM for 3D single molecule imaging and QZXZP3SM for any other type of imaging (including 2D single molecule imaging)
   
9. Click "Connect to Microscope"
   
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.

    ONI say that it will take the microscope 60 to 90 minutes for the microscope to reach 37°C and stabilise.

Putting a sample on the stage

1. Slide the magnets off the stage. DO NOT pluck them off as the strength of the magnet could damage the stage by pulling on it too hard.
   
2. Put a drop or two of immersion oil on the objective lens. Avoid getting bubbles in the oil as these will cause optical aberrations.
3. Make sure the bottom of the sample is clean, as dirt on the coverslip will severely impede focusing later on. If the coverslip has been used previously, make sure you clean it well with 70% ethanol.
4. Place your sample on the stage so that the area you want to image is above the objective lens pupil (e.g., a coverslip mounted on a slide, or one well of a chambered cover glass).

5. Drop the magnets into place on either side of the specimen so that it is held down onto the stage. There are six disc magnets in all, so the sample can be held down at several positions if necessary.
   

   There needs to be space for the magnets on either side of the specimen, so vessels like 35 mm dishes can’t be used with the magnets.

6. Once the specimen is in position, close the lid of the chamber and pull out the safety interlock slider, otherwise the imaging lasers won’t be able to switch on.

Focusing on the specimen

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.
   

   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).
   

   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 -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.
   

Setting acquisition conditions (for single colour 2D dSTORM)

The settings in the following section are appropriate for single-channel 2D dSTORM of a sample labelled with a far red dye like Alexa 647. Your sample or procedure may require other settings; such as an additional imaging channel, different laser wavelengths and powers, or 3D acquisition. If you need to use different settings, then you can either look for more information on the wiki or in ONI's knowledgebase (login required) or contact the light microscopy email: lmcb-lm-help@ucl.ac.uk.

1. Click the View button in the Acquisition Control area in the lower right of the UI to begin a live feed from the acquisition camera. You might need to scroll downwards in order to see the View and Acquire controls.
   
2. There will be two live images, which correspond to two simultaneous colour channels. In the top left next to the live images there is a small image with green and red rectangles. The two channels are split using a dichroic mirror and projected onto different regions of a single camera. The rectangles show the relative positions of the two channels on the camera chip.
   
3. For dSTORM using a far-red dye you only need Channel 1 (the one outlined in red), so you should untick Channel 0 in the Image Display Options on the left. This will prevent you from acquiring all the images from an unused channel. If you are using dyes emitting wavelengths shorter than far-red then you will need Channel 0.
   
4. The live camera image will only show noise until you illuminate the sample with a laser. Change the percentage laser power in the Light Control area by either clicking on the spinner, or typing in a value and pressing the Return key. It is best to use a lower power like 10% to avoid photobleaching while setting up your specimen. The A and B buttons allow you to create two laser illumination conditions, so you could have A set to 100% and B set to 10% and use the former for imaging and the latter for setting up. Click on the button above the laser (e.g. in this case the red 635 button) to switch on the illumination. The microscope will emit a high-pitched noise while the laser is operating. This is normal.
   
5. Generally speaking for single molecule techniques it is better to increase the contrast by operating the microscope in TIRF mode. In the Optical Control area on the right of the user interface, change the Target Illumination Angle from 0° to 53°.
   
6. Your sample will probably not be in focus initially. Normally the focus can be adjusted by changing the value in the Z: field (see area outlined in red in the Position Control image below), but if Z lock is active the focus cannot be changed this way. Instead you should adjust the Z offset, which offsets the focus position from the fixed Z Lock position.. Both the Z field and Z offset are highlighted with a red outline in the Position Control image below.
   
7. Once you're in focus turn off the laser by clicking its button in the light control area, so you don’t photobleach the specimen.

Creating a User Login

I recommend that you create a User Login. When you create a user login all your data will automatically be saved in your own folder, which can be useful when trying to differentiate your data from other users' data. If you don't login, all your files will be saved in the DEFAULT_USER folder.

1. Click the User login button in the top right of the user interface.
   
2. In the dialogue box that appears, type a Username and Password and click Create user.
3. Whenever you use the machine in future, simply click User login, type in your username and password, and click Login. Your images will be saved in a folder in the following location: C:\Data\your_username.

Acquisition Control

In the Acquisition Control area on the right at the bottom of the user interface you can define the exposure and number of images to be captured, as well as the naming scheme for the files. The Dataset tag is the name applied to a top-level folder that is created to hold all the image files captured during that session. A date stamp is automatically added to the folder name so there is no need to include the date in the name. Each individual acquisition during a session is named according to the text entered into the Acquisition tag field. If you've logged into your user login, all the acquired files and folders will be saved in your folder. The View button begins a live camera preview, which you can use while focusing or otherwise adjusting your acquisition settings. The A and B buttons allow you to create two sets of acquisition conditions.

To set up an acquisition for single-channel 2D dSTORM of a sample labelled with a far red dye like Alexa 647:

1. Set the exposure and number of images.
2. Define a dataset tag and an acquisition tag.
3. In the Light Control area, turn the laser power up to 100% (for dSTORM).
4. Click the red 635 button to start the laser illumination.
5. In the Acquisition Control area, press the Acquire button to begin capturing images.

The laser doesn't automatically come on when you press Acquire, which is why you need to turn the laser on in the Light Control area. Multiple Acquisition Setup automatically switches the laser on when acquisition starts, which is one reason why I prefer it.

Multiple Acquisition Setup

The settings in the following guide are appropriate for single-channel 2D dSTORM of a sample labelled with a far red dye like Alexa 647. Your sample or procedure may require other settings, such as an additional imaging channel, different laser wavelengths and powers, or 3D acquisition. If you need to use different settings, then you can either look for more information on the wiki or in ONI's knowledgebase (login required) or contact the light microscopy email: lmcb-lm-help@ucl.ac.uk.

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.
   

   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.
   
   

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.