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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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).
- 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.
- If you cannot see your sample you can try the following:
- Refocus on your sample in case it has drifted.
- 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.).
- Increase the laser power to get a better signal.
- 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.
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