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- Follow the QUICK START INSTRUCTIONS and the guide on GETTING STARTED - PUTTING A SPECIMEN ON AND LOCATING IT USING THE OCULAR for the N-STORM microscope. Make sure to select the Andor Zyla sCMOS camera during the software start-up procedure.
- At the bottom of the NIS-Elements software window select the Basic Widefield tab to go to the widefield acquisition layout.
- The lightpath for epifluorescence imaging should already be configured from the previous step in which you used the eyepiece of the microscope to focus on your sample. Therefore you should only have to click the L100 icon under Light Path in the TiPad to direct the light to the sCMOS camera. Also check that the LED (1) you would like to use to excite fluorescence of your sample is turned on and move the power slider to the desired value. Make sure that the Spectra shutter (2) is open the TIRF/EPI switch (3) is in the EPI position and the correct emission filter (4) for your fluorophore of interest is selected (blue, e.g. DAPI; green, e.g. GFP or Alexa Fluor 488; red, e.g. mCherry or Texas Red; SpectraX Quad for fast imaging of multiple colours with the same filter).
- Click on the Live Preview icon with the green play symbol at the top of the software to open a live preview window. The icon to the right of it with the red square pauses the preview. The camera icon allows you to take a snapshot of the current view. If you cannot see your sample try to re-focus on it and turn on the auto-scale option in the LUT panel to scale the dynamic range to your actual signal range. When you can see your sample you can also adjust the camera exposure time until you have a good signal, e.g. increase the exposure if your sample is too dim. Be aware that this will increase the time it will take to acquire your images. Alternatively you can increase the LED power by moving the power slider in the SpectraX panel. Again be careful not to increase the power too much as this may cause photobleaching. The example preview shown below was obtained using the Quad-band SpectraX emission filter and the 395, 470 and 555 nm LEDs at the same time. In the next step you will use pre-set or configure your own Optical Configurations for each colour channel that can then be used for advanced image acquisitions.
- In the OC (Optical Configuration) Panel you find some pre-configured optical configurations, e.g. for imaging DAPI, GFP or mCherry. The Optical Configurations are grouped for imaging with single emission band filters or the Quad-band filter for fast imaging with the SpectraX light source. There are also optical configurations available for using TIRF with laser excitation. Just click on the configuration you would like to use and the light path will be set up automatically. Press the Live Preview icon to start a preview and adjust the LED/laser power and camera settings if needed as described above. If you change the parameters of an optical configuration a red exclamation will appear in its button indicating this. To overwrite the optical configuration with the new settings click on the white arrow to the right of the exclamation mark. If you need an Optical Configuration that is not available in the pre-configured list you can create your own. Therefore start by configuring the lightpath for the experiment you are planning to do and check the Live Preview of your sample to see if it looks as desired. When happy with the settings click on the button displaying a blue plus and an objective in the OC Panel. This opens the New Optical Configuration window. Here you can name your new Optical Configuration, give it a colour LUT and configure which settings will be saved to the configuration, e.g. laser power or shutter position and which are not. Click Finish when done. A new button for your Optical Configuration will appear in the OC panel. You can drag and drop it in the menu where you want it or create a new one by pressing the menu plus button at the top of the OC Panel.