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  • Getting Started – SIM Imaging & Image Processing

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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.

    Image Added

  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.