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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.

 

  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. 




 

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