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Setting up a SIM experiment and recording beads for chromatic shift correction in multicolour SIM experiments

  1. For SIM imaging make sure to select the 63x oil objective. You can also use the 100x oil or the 63x water lens, but the 63x oil lens should be used preferentially.

  2. The first thing you should do every day when doing multicolour SIM imaging is to record a TetraSpeck (100 or 200 nm) bead stack to allow you to correct for chromatic shifts between the different colour channels. This is not required when you are doing one-colour imaging. Make sure to acquire your bead and sample images using the same objective lens and zoom settings.

  3. Therefore place the Channel Alignment bead sample (top drawer in the desk) on the microscope stage and focus on the beads as described in the section GETTING STARTED – PUTTING A SPECIMEN ON AND LOCATING IT USING THE OCULAR.

  4. Go to the Acquisition tab and in there expand Laser menu and check the lasers you want to use are turned on (available laser lines: 405, 488, 561 and 642 nm).

  5. Then expand the Light Path menu below the Laser menu.

  6. At the top you have the option to select between WF (widefield), SIM (Structured Illumination Microscopy) and Laser WF (laser widefield). Select SIM. In this acquisition mode TV2 (sCMOS camera) will be automatically selected as default camera and the 1.6 x Optavar lens and SIM lens and gratings (highlighted in red) will be inserted in the light path. You can still configure the light path manually as well, but here the recommended settings are shown.



  7. Configure tracks with the laser lines for excitation (click on the laser icon and select) and matching emission filters (see images below). The matching grating for each wavelength will be inserted in the light path automatically as soon as you select a laser (e.g. 28.0 µm at 488 nm; manually adjust the grating for fast imaging or when imaging thick specimen use larger grid at expense of resolution). New tracks can be added by pressing the +-button and removed by pressing the trash-button next to it.



  8. Go to the Channels menu and select different Look-Up-Tables (LUTs) for the tracks. By selecting each track you can see which laser it is configured for and adjust the laser power by moving the slider. Also adjust the exposure time for each colour channel here.



  9. Click on Live or Continuous to get a live preview of your specimen. Press Stop to stop the preview. Make sure to select only one track at a time and adjust the laser power and camera exposure time for each colour channel (if multiple tracks are selected the filter turret will constantly rotate). If the preview screen is dark, adjust the dynamic range of the camera by pressing min x max. If you still cannot see your sample check that all doors on the microscope enclosure are closed, since they are magnetically interlocked and will prevent the laser from turning on when open. Once you see an image re-focus if you have to and optimize the laser power and exposure time for each channel.



  10. Acquire a 4 µm Z-stack of the beads. Tick the Z-stack tick box on the top left of the screen to activate the Z-stack menu, which will appear under the Multidimensional Acquisition header. Setup a stack using the Centre mode (only available if Show All in the Z-stack menu is ticked, highlighted in blue frame). Therefore focus on the centre of the beads, stop the live preview and press Centre. Turn on all laser lines in the Channels menu, then back in the Z-stack menu click smallest to optimize the step size of your stack. By changing the number of slices adjust the stack range to ~ 4 µm.

       

  11. Make sure that all tracks you want to image are active, then press Start Experiment to acquire your bead calibration file.



  12. Once you have acquired the beads you can start imaging your sample of interested using the instructions for Setting up a SIM experiment and chromatic shift correction in multicolour SIM experiments 1-9 (or 1-11 if you want to record a Z-stack of your sample). You can record 2D or 3D (z-stack) datasets by selecting Start Experiment, just make sure that the calibration beads were recorded using the same frame size and using all colour channels that you are looking at in your sample.

A typical example image of SIM raw data before Structured Illumination processing often shows the grating pattern that was projected onto the sample during imaging.


Processing SIM data

  1. Images can be taken to the ZEN analysis PC in room 2.19A for processing.

  2. To obtain a SIM image from your raw data go to the Processing tab (1) in the ZEN Black software. Select Structured Illumination and a dropdown menu opens. Here select Structured Illumination again (2). A menu window opens in which you can set up the Method Parameters (3). You need to do the Structured Illumination processing step for all of your sample images and the calibration bead stack.



  3. Under Method Parameters press Select (1) to select the currently active image for the processing. The slices slider below the Input allows you to select, which part of a z-stack you want to process. Then choose between the Automatic and Manual processing mode in the dropdown menu (2). If the Automatic mode does not deliver the desired result e.g. produces processing artefacts in the images try switching to the manual mode to optimize the processing parameters. For the Output you can select between a SR-SIM (super-resolution SIM), Wide Field (Pseudo widefield) and DCV (deconvolved) image (3).



  4. If using Automatic processing (recommended): Just select between 2D (single slice image) and 3D (Z-stack), what output you want (SR-SIM, DCV or Wide Field) and press Apply at the top of the screen to start the image processing. It is also possible to batch process files. The processing can take some time (≥ 30 min) depending on the size of your image file. 



  5. If using Manual processing: Select Manual (1) in the Processing dropdown menu to open the Manual processing menu (2) and then the 2D or 3D mode depending on whether you would like to process a single slice image or a Z-stack.

  6. In Settings for Channel (a) choose between processing all channels or just a specific one. Below that you can select from a range of SIM processing parameters including Max. Isotrop (b), Auto Noise Filter (c) and SR Frequency Weighting (d). You will need to try what will work best for your images. Selecting Max. Isotropy (b) will slightly blur lines in the FFT. Untick the Auto Noise Filter checkbox (c) to get access to the slider with which you can adjust the (Wiener) filter level. The default setting is -6, but you may get better results adjusting this value depending on the Signal-to-Noise ratio of your sample. A typical value would be between -5 to -6. Check for unwanted artifacts following processing with each parameters and try to remove them by optimizing the filter settings. Keep the slider for the image Frequency Weighting (d) at 1.0. Baseline Cut is the default baseline setting, but it can be useful to use Baseline Shifted to positively shift the image if Noise filtering was applied and introduced negative pixel values in the image.



  7. Select SR-SIM or any other format you want from the Output dropdown menu (e).

  8. Chose Theoretical from the PSF dropdown menu (f).

  9. Press Apply at the top of the screen to start the SIM processing.

  10. Save the SIM image in .czi file format. ZEN will automatically append “Structured Illumination” to the filename of the output image.


Correcting SIM images for chromatic shifts – Channel alignment

This analysis step is only required for datasets containing more than one color channel.

Obtaining the channel alignment transformation table from your bead data

  1. In the Processing tab select the Channel Alignment tool from the list of Methods.



  2. In the Method Parameters tab select the file that you obtained from the Structured Illumination processing of your bead stack by opening the file in ZEN and clicking Select. The filename of the selected dataset will appear next to the button.

  3. In the Channel Alignment options make sure Fit is checked and select Affine from the dropdown menu (not Lateral).

  4. Press Apply near the top of the screen to start the processing (takes some time to run). 



  5. In the resulting image all colour channels should be properly aligned now. Save the image file as .czi and more importantly the transformation table on the left as a bin file by clicking the Save-button. 





Applying the channel alignment to your SIM images

  1. Run the Structured Illumination analysis as described in the section Processing SIM data above for your sample image.

  2. Save the output file.

  3. In the Processing tab select the Channel Alignment tool from the list of Methods.

  4. In the Method Parameters tab select the Structured Illumination output image of your sample that you want to correct for chromatic aberrations by opening it in ZEN and clicking Select. The filename of the selected dataset will appear next to the button.



  5. Make sure to untick Fit this time and select Affine in the dropdown menu.

  6. Then click on the Load-button under the empty transformation table, select the bin file you saved earlier and press Open. Now the table should display the values for the colour shifts between channels.

  7. In the table below make sure the wavelength channel IDs correspond to the wavelengths of the channels in the transformation table above.

  8. Press Apply to apply the transformation table to your sample image.

  9. Save the resulting file in .czi format. ZEN will automatically append “Channel Alignment” to the output filename.



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