Page Comparison

...

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. 
   Image Added
   
   Image Added
   
   
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.
   
   Image Added
   
   
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…