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  • Getting Started - Multiparameter Widefield Imaging
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  1. 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.



  2. At the bottom of the NIS-Elements software window select the Basic Widefield tab to go to the widefield acquisition layout.



  3. 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). 



  4. 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. 



  5. 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.



  6. In the ND Acquisition panel you can configure advanced image acquisitions including sequential multicolour imaging, time-series or multipoint acquisitions. At the top of the panel you can configure auto-save settings (1) by ticking the Save to File checkbox and then selecting the Path where you want to save your data and entering a file name. Alternatively you can manually save your data after the acquisition by highlighting the image file and navigating to File -> Save as…. The default file format of the NIS-Elements software is .nd2, which contains the image files including the metadata of your experiments. Below the auto-save options are five tabs (2) that allow you to set up a time series (Time), a multipoint acquisition (XY), multicolour sequential imaging (λ), a Z-stack acquisition (Z) or a tiled overview image (Large Image). The different acquisition modes can also be combined. Just tick the checkbox for the tabs you want to use. You can then change the parameters in each tab. This will be described for each tab in more detail in the following. You can also change the order of the tabs to change order in which the acquisition steps are carried out. 



  7. To acquire a multicolour channel image tick the checkbox on the λ-tab to activate it. Then go to the Optical Configurations column in the table below and select the ones that you would like to use from the list of pre-configured Optical Configurations. Add more channels (rows) by clicking the Add-button above the table or by ticking the checkbox at the beginning of the next available row. The Name and Color columns will be filled in automatically based on the Optical Configurations you chose. To set the Focus Offset you need to switch on the PSF (optional). The order of the channels can be changed by highlighting a channel with the mouse and clicking on the up/down-arrows above the table to change its position. Individual channels can be deleted by highlighting them and clicking on the X-icon. All channels can be cleared by pressing the Delete All icon on the far right above the table. Tick the Close Active Shutter During Filter Change checkbox under the table to protect your sample from prolonged exposure to excitation light. When all channels are set up press the Run Now button to start the image acquisition.



  8. To acquire a time series tick the checkbox on the Time-tab to activate it. Highlight the first row in the Time schedule table to create a Phase. Enter in which time interval you would like to acquire your images in the Interval column. In the Duration column you can configure the total time to acquire images for or alternatively enter the number of times to repeat the set interval in the Loops column. Adding Phases allows you to record time-series with different Intervals, e.g. you could set up a Phase 1 in which you record an image every 30 s for 5 min and then a Phase 2 in which images are taken at a longer interval such as every 5 min for 2 h.  If multiple Phases have been configured you can use the up/down-arrow icons above the table to swap them around. Highlight a Phase and press the X-con to delete it. Ticking the Close Active Shutter When Idle checkbox prevents prolonged exposure to the excitation light when no images are recorded. If images need to be acquired as fast as possible it is recommend not to tick this. Press Run Now to start the time-series acquisition.



  9. To acquire images a multiple position in your sample tick the checkbox on the XY-tab to activate it. Turn on a Live Preview and use the XY-stage controller to navigate to the first position in your sample of which you would like to take an image. Focus on the sample using the normal focus wheel or turn on the PSF and adjust the PFS Offset. Highlight the first row in the Points table and press the Add-button to add the position to the points list. Move to the next position in your sample and repeat the process. You can add a many points as you like this way. The order of points can be swapped by highlighting the point and using the up/down-arrows to change its list position. To delete an individual point highlight it and press the X-icon above the Points table. To clear the entire list click the Delete All icon next to the X-icon. If you want the Z-position of your points to be included in the acquisition rather than just the XY you need to tick the Include Z checkbox under the Points table. If you tick the Move Stage to Selected Point checkbox above the table the stage will move to a point automatically when you click on it. For the Z-device you can chose between using the Ti ZDrive (Nikon motorized stage) and the Mad City Labs piezo drive, which is more accurate. Checking the Close Active Shutter During Stage Movement checkbox can prevent prolonged exposure of your sample to excitation light and hence photobleaching. Press Run Now to start the multiposition acquisition.



  10. To acquire a Z-stack tick the checkbox on the Z-tab to activate it. There are different ways to set up a Z-stack available. You can configure a Bottom and Top position to set a range and define a step size by selecting the Bottom/Top-icon. Alternatively select the Symmetric or Asymmetric mode icon to configure a Z-stack by setting up a range around a Home position (a certain focal plane). The easiest way to set up a Z-stack is to turn on the Live Preview and use the Z-focus controller to move to the top position and press the Top button, then move to the bottom position and confirm it by pressing on Bottom. If you are using the Home position mode focus where you would like to set the home position, then click the Home button. You can chose between using the Ti ZDrive (Nikon motorized stage) and the Mad City Labs piezo drive, which is more accurate to step through the sample. It is also possible to include a TIRF position into the stack. Press Run Now to start the Z-stack acquisition.




  11. To acquire a large tiled image tick the checkbox on the Large Image-tab to activate it. Define the scan area by putting in a value for the number of fields you want your image to be wide and high. Decide if you would like the fields to be automatically stitched together by the software or kept separated under Stitching. Press Run Now to start the Large Image acquisition.



  12. When pressing Run Now a ND Progress window will open displaying the Experiment overall progress, an estimate of the time that has already elapsed and the remaining time it will take to complete the image acquisition. You also have the option to Pause the experiment and resume it later, Finish the acquisition manually without losing the data that has already been acquired or Abort the experiment (all data will be lost).



  13. If you have not turned on the auto-save option save your acquired images by highlighting the image in NIS-elements and navigating to File - > Save as… option. It is recommended that you save all file in the .nd2 format.

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