Getting Started - STORM Imaging & Analysis

In the following is described how to configure the Nikon N-STORM microscope for STochastic Optical Reconstruction Microscopy (STORM). If you require more information on the preparation of biological specimens for STORM imaging please refer to the sample preparation links on the main Nikon N-STORM page. The instructions are specifically for STORM imaging, so if you are looking for information on starting up the microscope please go to the QUICK START INSTRUCTIONS. For information about putting a specimen on and focusing on it go to GETTING STARTED - PUTTING A SPECIMEN ON AND LOCATING IT USING THE OCULAR.

STORM Imaging

1. These instructions assume that you have already switched on all components of the microscope and placed your specimen on the microscope stage and focused on it using the ocular. If focusing through the ocular is not possible e.g. because your sample is labelled with a far red dye, you can also do this using the live preview mode in the NIS-Elements software.
   
   
2. Remember to select Andor with N-STORM in the camera driver selection dialog when starting up the software, so the Andor iXon DU-897 EMCCD camera required for STORM acquisitions will be available in the software
   
   
3. At the bottom left of the NIS-Elements software navigate to the N-STORM acquisition tab, which opens a software layout containing all controls required for the acquisition of STORM images.
   
   
   
   
   
4. Now you need to configure the light path and camera settings for imaging. Therefore go to the Ti Pad and select the CFI Apo TIRF 100x oil (NA 1.49) objective (remember to use immersion oil) (1) and direct the light to the right camera port (R100, Andor iXon EMCCD) (2). Then at first select EPI and set the TIRF slider roughly to the centre position (3). You are now in normal widefield illumination mode which will help you to focus on your sample in the live preview. You will adjust the illumination angle for imaging in later. Last select the correct filter cube (4) for STORM imaging. You can pick between the C-STORM quad filter (C-STO) optimised for emission detection when imaging with 488, 561 or 647 nm excitation or the single band STORM (N-STO) filter optimised for use with the 647 nm excitation laser. 
   
   
   
   
5. Navigate to the N-STORM with Agilent Acquisition tab. If the STORM acquisition controls are not displayed right click anywhere on the screen, navigate to Acquisition controls and select N-STORM with Agilent Acquisition. In the same way you can also open the N-STORM Launch pad.
   
   
6. In the N-STORM with Agilent Acquisition tab select the laser line you would like to use for your experiment (1) (e.g. 561 or 647 nm) and move the power slider to a power high enough percentage power (2) to visualize your sample (e.g. ~ 2 - 10 %). Open the Agilent shutter below the lasers (3) and make sure that the ILOCK button near the top of the software (4) is selected to disable the laser interlock system (Note: also make sure that the interlock lid is placed on the sample stage).
   
   
   
   
   
7. To preview your sample press the live preview button (1) at the top of the NIS-Elements software (if you have multiple cameras enabled make sure to press the Live button next to the camera you want to use, here the Andor iXon DU-897 EMCCD) and a window with a live image (2) will open. Shown in the example below is a widefield image of GattaPaint 80RG nanorulers.
   
   
   
   
8. If you cannot see your sample you can try the following:
   1. Refocus on your sample in case it has drifted.
   2. If you are in focus but only have a low signal increase the camera exposure time and/or EM Gain Multiplier until you get a good image (see 9.).
   3. Increase the laser power to get a better signal.
   4. Adjust the Look-Up-Table (LUT) histogram so that you are only looking at the part of the dynamic range (grey levels) where you actually have a fluorescence signal. This can be adjusted using the dynamic range scale buttons in the LUT tab highlighted within the red box.
   
   
   
   

9. The camera acquisition setting can be adjusted in the DU-897 Settings tab. The most important settings to adjust are the Auto Exposure and the EM Gain Multiplier. Adjust them until you have a good quality image. Example settings for STORM imaging are:

   • Format for Live: No Binning

   • Desired Temperature: -70°C (allow some time to cool down after switching on the camera)

   • Conversion Gain: Maximum available value (5.1x)

   • EM Gain Multiplier: ~ 30 - 100

   • Readout mode: EM Gain 10 MHz 14-bit

   • Exposure time: e.g. ~ 30 - 50 ms or 1 frame, but this should be adjusted depending on the sample brightness

   • Format for Capture: No Binning

   
   
   
   

10. To get a sufficient laser power intensity at your sample for STORM imaging insert the STORM slider (A, B) and λ-slider (C) at the back of the microscope and make sure that the ND filters (C) are out of the lightpath (please refer to the picture part B and C for the correct STORM slider and filter configurations).
    
    
    
    

11. Check that the Optovar lens at the front of the microscope stand is set to 1x rather than 1.5x magnification.

    1.5x zoom lens

    From Nikon's N-STORM Super-resolution Microscope Operation Manual: "STORM cannot offer the guaranteed level of accuracy unless the recommended configuration and settings are used. Note that, if a 1.5x intermediate magnification lens or a 60x objective is used, STORM does not function properly, because the configuration is not the recommended one."

12. Make sure to configure the camera ROI to a maximum size of 256 x 256 pixels. Therefore click on the little white arrow next to the ROI button in the image preview window and select Define ROI (1). A new dialog (Rectangle) opens in which you should enter 256 for the Height and the Width value (or smaller) and the ROI will be displayed in the live preview window (2). Press OK to confirm your selection (3). The preview will now display the selected ROI.
    
    
    
    
13. Change from EPI to the TIRF illumination mode and adjust the TIRF angle by moving the TIRF slider until your sample looks the brightest when focused near the coverslip and is evenly illuminated. The exact position may vary depending on your sample. The slider can be moved by dragging it with the mouse or click the mouse icon to adjust the TIRF angle using the mouse track wheel. The track wheel sensitivity can be set to Coarse, Fine or Extra Fine.
    
    
    
    
14. Make sure that the cylindrical lens is not inserted in the lightpath for 2D image acquisitions, but pushed in for 3D STORM acquisitions. The cylindrical lens can be found on the right hand site of the microscope stand behind the focus wheel in the camera lightpath.
    
    
15. Pause the preview by pressing the Stop preview button (1) and close the Agilent shutter (2).
    
    
    
    

16. Now that you have set up the lightpath, and hopefully obtained a good preview of your sample you are ready to configure your STORM experiment settings. The STORM acquisition panel consist of 2 main parts as shown below: the N-STORM Launch Pad and the N-STORM with Agilent Acquisition. By pressing the buttons 1 – 4 in the N-STORM Launch Pad you can open the N-STORM with Agilent Acquisition pad (1), a live STORM reconstruction preview window (2), a live STORM Localization Counts window (3) and the STORM Analysis window (4).
    
    
    
    
    

17. Select the excitation laser for your experiment by clicking on the button with the wavelength you would like to use (here 561 nm) (5) at the top of the N-STORM with Agilent Acquisition Pad.
    
    
18. Tick the matching STORM configuration below (same excitation wavelength/Reporter probe) to turn on spontaneous activation (6). In the spontaneous activation mode excitation and imaging are achieved using a single laser (only the Reporter Probe is required).  For continuous activation additionally tick the activation laser (Activation Probe, usually 405 nm) underneath the Reporter Probe.
    
    
19. If the desired laser combination is not available or you need to adjust the number of Reporter and Activation Frames click the Settings button to go to the Agilent Acquisition Settings window. For spontaneous continuous activation set Activation Frames to 0 and Reporter Frames to 1 under Cycles. For continuous activation a common setting is 1 Activation and 3 Reporter Frames, but it will depend on the dye you are using. The settings in Channel 1, 2, 3 determine what laser combinations are displayed in the N-STORM with Agilent Acquisition (6). Change them here if needed for your experiment.
    
    
20. In the field Period Count (7) enter the total number of frames for which you would like to acquire your images. Usually the Period count is set somewhere in the range 5,000 – 20,000.
    
    
21. It is possible to acquire 2D or 3D data sets, therefore select 2D or 3D STORM (8). If you selected 3D STORM make sure to insert the cylindrical lens into the lightpath which is located on the right hand site of the microscope stand behind the focus wheel in the camera lightpath and also check if a 3D calibration file is available for the lightpath settings you are using (there should be a folder with calibration files on the desktop). If there is no 3D calibration file available contact the light microscopy core staff to get help with recording one. Calibration files needs to be recorded whenever major changes are done to the light path, such as changing emission filters or realigning the lasers.
    
    
22. Now select a Path and set a File Name for auto-saving your data (9). The files are saved in the .nd2 NIS-Elements format containing all image acquisition metadata.
    
    
23. To make sure that the focus is maintained stable throughout the experiment turn on the Perfect Focus System (PSF) in the software within the TiPad or on the front of the microscope. The main ON button will turn green and if the PFS is available the little LED below it will turn orange (NOTE: with the 100x oil lens the PSF is only available for samples mounted on a glass coverslip or glass-bottom chamber that are immersed in liquid medium, in some cases it may also work with samples mounted in Vectashield). Adjust the Offset using the PSF Offset Controller until your sample is in focus. The normal focus wheel is not responsive when the PSF is turned on.
    
    
    
    
24. Close the Agilent laser shutter.
    
    
25. 2D STORM:
    1. For a spontaneous 2D STORM experiment bring the excitation laser power up to 100% and press Run Now (10) to start the image acquisition.
    2. For a continuous 2D STORM experiment make sure to select an activation probe as described above and turn on the corresponding activation laser from the list of laser lines. Initially set it to a low power (0.3 – 0.5 %). Then either manually increase the activation laser power throughout the experiment as required or click the Auto LP button underneath the laser lines and enter a maximum value to let the software control the activation laser power (it will try adjust the power to ensure that the number of molecules detected per frame will remain constant throughout the acquisition). To check if the activation is working turn on the Live Preview with the excitation and activation laser selected for ~ 1s, then deselect the activation laser again (usually 405 nm). The live image should become bright and then return to the previous state. If you are using multiple activation lasers check this for all of them. Press Run Now (10) to start the continuous STORM acquisition.
       
       
26. 3D STORM:
    1. For a spontaneous 3D STORM experiment check that 3D STORM is selected and the cylindrical lens in the EMCCD camera lightpath is inserted. Turn in the PSF. Bring the excitation laser power up to 100% and press Run Now (10). You will be prompted to focus on the specimen. Do this using the PSF Offset Controller and press OK. Then focus on the boundary surface of the coverslip and your specimen. Press OK again after doing so and the stage will automatically return to the set focus plane inside your specimen. Check if this is correct, then press OK to start the image acquisition.
    2. For a continuous 3D STORM experiment follow the instructions in 25b for setting up a continuous 2D STORM experiment. Before pressing Run Now follow the instructions in 26a to configure the 3D STORM lightpath and start the image acquisition.
       
       
27. As soon as the image acquisition has started a widefield image live preview (1) and a progress dialogue (2) will be displayed. From the N-STORM Launch Pad (3) you can manually open the live STORM reconstruction preview window (4) and the live STORM Localization Counts window (5) to get an idea of whether you are collecting sufficient molecules per frame. The density of bright spots in your sample should not be too low or too high (overlapping molecules). Approximately 100 – 200 bright spots per frame are ideal. This can be adjusted by manually changing the power of the activation laser. If AutoLP is selected the software will adjust this automatically. 
    
    
    
    

28. A notification saying “STORM Analysis Completed” will pop up when the STORM image acquisition has finished. Your images will have auto-saved and you can process and reconstruct your super-resolution data directly in NIS-Elements or using an external software package, e.g. ImageJ.

    

STORM Image Analysis

1. Go to the Launch Pad and click the N-STORM Analysis button to open an empty N-STROM Analysis window. In the new window click on the folder icon, locate the file you want to process and click Open to display the STORM dataset.
   
   
   
   
2. The widefield images of all frames you have acquired will be displayed. Use the slider at the bottom of the image window to navigate through the different time points. You can right click on the slider at any point to open a menu that allows you to set a start and/or an end point for the image processing, e.g. this can be useful if the first few frames are too bright to localize any molecules and you therefore do not want to include them in the analysis.
   
   
   
   
3. Press the Peak Statistics button on the right and hover over the background to get an idea of the background intensity in your sample. Do this for a few frames and note down the average value. Note that the peak statistics option is not available when Auto Minimum Height is selected in the Identification Settings.
   
   
   
   
4. Click on the Identification Settings icon (or the STORM Analysis icon and then Identification settings) to go to the settings menu and configure the Minimum Height with the value that you measured in the previous step. Alternatively tick the Auto Minimum Height checkbox to allow the software to determine the minimum intensity value for the image processing. The Maximum Height is automatically set to 65,536, which corresponds to the number of grey levels of the 16-bit EMCCD camera is used for the imaging. The CCD Baseline value is 100. Select 3D if it is a 3D dataset otherwise leave the checkbox unticked. By clicking the double arrow button you get to an advanced menu where you can adjust further Identification settings like the Minimum Width (i.e. 200) and the 2D or 3D Maximum Width (i.e. 400 or 700 respectively). Set the Fit Width (nm) to 300, the Max. Axial Ratio to 1.3 for 2D data and 2.5 for 3D data and the Max. Displacement (pix) to 1. Click OK after you have set up all Identification Settings. 
   
   
   
   
   
5. Click on the STORM Analysis icon. In the window tick the Drift Correction checkbox. After configuring the Identification settings enter the number of frames for which you would like to run a test analysis in the Periods field (e.g. only runs for the currently selected frame). Click Test. A test analysis will be run for the selected frames and when done a dialogue saying “STORM Test Analysis Completed” will open. Detected molecules will be highlighted with a yellow box around them. Check whether all molecules have been identified correctly and when you are happy with the result click on the STORM Analysis icon again and press Start to run the analysis of the entire dataset. If not, adjust the Identification settings and repeat the test until all molecules are identified correctly then run the complete analysis.
   
   
   
   
   
   
   
   
6. When the Analysis is complete the molecule localization information for all molecules is saved in binary format as a Molecule list including the Drift correction information alongside the original ND2 file. You can also manually save a .txt file of the molecule list by selecting Save as.. and scrolling to .txt in the Save file as … dropdown menu. If you are moving the file to a different storage place make sure to keep them together as otherwise they may become unreadable.
   
   
7. To save your images including the molecule list click on the Floppy Disk icon at the top of the analysis window.
   
   
8. By clicking the Camera icon you can take a snapshot of the currently opened image and save it in a different file format e.g. tif (NOTE: Metadata may be lost when saving your image in this way).
   
   
9. Additionally you can click the Filter icon after the analysis has been completed and apply some filters to the displayed molecules, e.g. the radius filter can help to remove low density detections in the background (looks at distance between molecules).
   
   
   
   
10. The icons on the left of the N-STORM Analysis image window allow you to configure how your image is being displayed. To see what the icons do have a look at the picture in step 4.
    
    
11. If you do not want to use NIS-Elements to reconstruct your STORM images you can also use a range of freely available software and plugins, such as Thunderstorm in ImageJ. For more information about alternative STORM analysis software please have a look here…