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Apotome Plus

This module offers a novel reconstruction algorithm for Apotome 2 and 3. Based on an iterative joint SIM reconstruction, it increases SNR, lateral and axial resolution compared to predecessor algorithms.

Apotome Plus processes 3D z-stacks and runs on ZEN 3.11 and higher. Note that a separate workstation running ZEN desk is recommended for processing during acquisition, as the workstation requirements are significantly higher than for previous methods. Specific guidelines and recommended configurations can be found in the price list.

Apotome Plus (defaults)

This method allows you to select two different algorithms for Apotome Plus, without any further settings.

Parameter

Description

Good, Medium Speed (Joint Fast Iterative)

Uses an algorithm based on Deconvolution methods for structured illumination microscopy, with some enhancements as described in the technical note. It is faster and less memory intensive, and also purely using the image formation model.

Excellent, Slow (Joint Constrained Iterative)

Uses an algorithm based on the Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy, but modified to allow for joint reconstruction and with enhancements as described in the technical note. It offers increased robustness to noise and mismatch between the theoretical and real PSF.

Using Apotome Plus in Direct Processing

Apotome Plus can be used in Direct Processing. For general information, see Direct Processing.

  1. If you are using Direct Processing on different computers, you have connected acquisition and processing computer, see Connecting Acquisition Computer and Processing Computer.
  2. To ensure that the processing computer reads incoming files and starts the processing, on the Applications tab, in the Direct Processing tool, you have clicked Start Receiving. This is usually active by default.
  3. On the Acquisition tab, Direct Processing is activated. This activates the Auto Save tool as well.
  4. Depending on your settings, you have defined the folder where the acquired images are stored in the Direct Processing or the Auto Save tool. Use a folder to which the processing computer has access. For information about sharing a folder, see Sharing a Folder for Direct Processing.
  5. On the Acquisition tab, you have set up your experiment for Apotome acquisition.
  6. If you want to use advanced settings created with the Apotome Plus (adjustable) function, you have the settings available, see also Creating Apotome Plus Settings.
  1. On the Acquisition tab, open the Direct Processing tool.
  2. The tool parameters are displayed. In the processing pipeline, the first block is selected automatically
  3. If the function is not preselected, go to the Processing Function dropdown list and select Apotome Plus.
  4. The parameters are displayed.
  5. Select the algorithm you want to use. If you want to use an advanced settings file you have created with the function Apotome Plus (adjustable), activate Use Advanced Settings.
  6. A dropdown list is displayed under Load Setting created in the Apotome Plus function.
  7. From the dropdown list, select your advanced settings for Apotome Plus.
    Note: Currently Direct Processing supports settings configured on the Apotome Plus tab of the image processing function Apotome Plus (adjustable) and some parameters of the PSF Settings tab. Especially parameters that rely on external data (like using external PSF) are not possible with Direct Processing.
  8. Click Start Experiment to run the experiment. Note: You can pause the processing. If you stop the experiment, requests that have been sent earlier by the acquisition computer are not processed. However, already processed images will be retained.
  9. The images are stored in the folder you have defined in the Auto Save or Direct Processing tool. When you abort the acquisition, the remote processing will not take place. In case you have set up several processing functions, only the acquired image and the final output image are stored.
  10. The processing computer reads incoming files and starts the processing. The path to the selected folder, the currently processed image as well as the images to be processed are displayed in the Direct Processing tool. The processed image is saved to the same folder specified in the Direct Processing tool. If the image name already exists in this folder, the new file is saved under a new name <oldName>-02.czi.
  11. To cancel the processing on the processing computer, on the Applications tab, in the Direct Processing tool, click Cancel Processing.
  1. Once processing is finished, you are notified on the acquisition PC and can open and view the acquired image as well as the processed image. This should be done on the processing computer, so that you can immediately start a new experiment on the acquisition computer. However, you can also automatically open the processed image on the acquisition PC with the respective setting in the Direct Processing tool on the Acquisition tab.
  2. Information about Direct Processing (e.g. the duration) is available on the Info view tab of the processed image.

Creating Apotome Plus Settings

You can create settings for Apotome Plus which can be saved, exported, and imported.

  1. You have opened an image based on which you want to define the processing settings.
  1. Open the Processing tab and select the method Apotome Plus (adjustable).
  2. The parameters are displayed in the Parameters window.
  3. In the Input window, select the desired image for Apotome Plus.
    Note: If you use the settings for Direct Processing, use a test image acquired with the identical experiment settings you will be using when running the experiment with Direct Processing.
  4. In the Parameters window, activate Show All (if it is not already activated) and click the context menu button gray camera icon on dark square background
  5. A dropdown list is displayed.
  6. Select New.
  7. The text field gets editable.
  8. Enter a name for your settings and press Enter on your keyboard, or click on the save button small floppy disk icon with metallic top.
  9. You have defined a name for the setting.
  10. Configure your settings in the Apotome Plus or PSF settings tab, see also Apotome Plus Tab and PSF Settings Tab Apotome Plus (adjustable). Note that if you want to use this setting in Direct Processing, some parameter settings of the PSF Settings tab are not supported/possible there, especially parameters that rely on external data (like using external PSF).
  11. Click gray camera icon on dark square background and select Save.
  1. You have now created and saved an Apotome Plus setting. You can load this setting into the Direct Processing tool to use it for a Direct Processing experiment.

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