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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.

Apotome Plus (adjustable)

This method allows you to use and individually configure two different algorithms for Apotome Plus. Two tabs are available for detailed configuration:

PSF Settings Tab Apotome Plus (adjustable)

All key parameters for generating a theoretically calculated Point Spread Function (PSF) are displayed on this tab.

Usually, images (with file type *.czi) that have been acquired with ZEN automatically contain all microscope parameters, meaning that you do not have to configure any settings on this tab. Therefore, most parameters are grayed out in the display. It is possible, however, that as a result of an incorrect microscope configuration values may not be present or may be incorrect. You can change them here. The correction of spherical aberration can also be set here.

Parameter

Description

Get PSF Parameters from Input Image

Activated: The parameters of the PSF are taken directly from the input image and the respective parameters are displayed and grayed out (not editable). This is the default setting.

Deactivated: Enables you to manually change the PSF parameters.

Advanced Section

Only visible if the Show All mode is activated.

Parameter

Description

Phase Ring

If you have acquired a fluorescence image using a phase contrast objective, the phase ring present in the objective is entered here. This setting has significant effects on the theoretical Point Spread Function (PSF).

PSF Generation

Selects the model for calculating the PSF.

–

Scalar Theory

The wave vectors of the light are interpreted as electrical field = intensity and simply added. This method is fast and is sufficient in most cases (default setting).

–

Vectorial Theory

The wave vectors are added geometrically. However, the calculation takes considerably longer.

Z-Stack

This field can only be changed if it was not possible to define this parameter during acquisition, e.g. because the microscope type was unknown. It describes the direction in which the z-stack was acquired. Note that this setting is only relevant, if you are using the spherical aberration correction.

–

Descending

The z-stack descends away from the objective.

–

Ascending

The z-stack ascends towards the objective.

Aberration Correction Section

Only visible if the Show All mode is activated.

Here you can select whether you want spherical aberration to be taken into account and corrected during the calculation of the PSF. As with the other PSF parameters, most values are extracted automatically from the information about the microscope that is saved with the image during acquisition. The input option is therefore inactive.

Parameter

Description

Enable Correction

Activated: Uses the correction function. All options are active and can be edited.

Embedding Medium

Select the used embedding medium.

Refractive Index

Displays the refractive index of the selected embedding medium. Enter the appropriate refractive index if you are using a different embedding medium.

Manufacturer

Displays the manufacturer, if known.

Depth Variance

When Aberration correction is activated, it is also possible to enable the creation of depth variant PSFs. This method allows for dramatic improvements in image restoration of thicker samples by creating axially variant theoretical PSFs as a function of the distance to the coverslip and the refractive index of the mounting medium.

Activated: Uses depth variant aberration correction. In the spin box edit field you can define how many PSFs should be generated. The more PSFs you create, the better the results, but selecting many PSFs increases the processing time. You should choose at least 3 PSFs.

In the dropdown list you can choose between the PCA method (Primary Component Analysis, M. Arigovindan et al., 2005, IEEE Transactions on Image Processing 14. nr. 4 p.450ff) which is best suited for constrained iterative and fast iterative method and the Strata method (Myneni and Preza, Frontiers in Optics 2009, Optical Society of America, paper CThC4.), which is best for regularized inverse filter and Richardson Lucy iterative deconvolution.

Distance to Cover Slip

Displays the distance of the acquired structure from the side of the cover slip facing the embedding medium. Half the height of the z-stack is assumed as the initial value for the distance from the cover slip. The value can be corrected if this distance is known. If possible, this distance should be measured.

Note: Use Ortho View and the Distance Measurement option to define the distance of the sample to the coverslip. It is also important to estimate the position of the glass/embedding medium interface as precise as possible. If the z-stack extends into the coverslip, the determined range of the stack which reaches into the glass should be entered as a negative value. Example: Z-stack is 26 µm thick, glass/medium interface is positioned at 9 µm distance from the first plane of the stack. Resulting value for Distance to cover slip: - 9.0 µm.

Cover Slip Type

Commercially available cover slips are divided into different groups depending on their thickness (0, 1, 1.5 and 2), which you can select from the dropdown list. Cover slips of the 1.5 type have an average thickness of 170 µm. In some cases, however, the actual values can vary greatly depending on the manufacturer. For best results the use of cover slips with a guaranteed thickness of 170 µm is recommended. Values that deviate from this can be entered directly in the input field.

Cover Slip Ref. Index

Selects the material that the cover slip is made of. The corresponding refractive index is displayed in the input field next to it.

Working Distance

Displays the working distance of the objective (i.e. the distance between the front lens and the side of the cover slip facing the objective). The working distance of the objective is determined automatically from the objective information, provided that the objective was selected correctly in the MTB 2011 Configuration program. You can, however, also enter the value manually.

Override

Only active if Enable Correction is activated.
Overrides the value. To reset the values, click Reset.

Channel Specific Section

In this section you find all settings that are channel specific. This means that they can be configured differently for each channel.

Parameter

Description

Illumination

Displays the excitation wavelength for the channel dye [in nm] by using the peak value of the emission spectrum. The color field corresponds to the wavelength (as far as possible).

Detection

Displays the peak value of the emission wavelength for the channel dye. The color corresponds to the wavelength (as far as possible).

Sampling Lateral

Depends on the geometric pixel scaling in the X/Y direction and displays the extent of the oversampling according to the Nyquist criterion. The value should be close to 2 or greater in order to achieve good results during DCV. As, in the case of widefield microscopes, this value is generally determined by the objective, the camera adapter used and the camera itself, it can only be influenced by the use of an Optovar. With confocal systems, the zoom can be set to match this criterion.

Sampling Axial

Depends on the geometric pixel scaling in the Z direction and displays the extent of the oversampling according to the Nyquist criterion. The value should be at least 2 or greater in order to achieve good results during DCV. This value is determined by the increment of the focus drive during acquisition of Z-stacks and can therefore be changed easily.

Microscope Info Section

Displays advanced microscope information that influences the form of the PSF in a channel-dependent way:

Parameter

Description

Illumination

Selects the illumination method with which the data set has been acquired. In the event that a Conventional Microscope has been entered under the microscope parameters, the following options are available here: Epifluorescence, Multiphoton Excitation and Transmitted Light. In the case of confocal microscopes, Epifluorescence is the only option.

Image Formation

Displays whether the imaging was incoherent (Conventional Microscope) or coherent (Laser Scanning Microscope).

Lateral Resolution

Displays the lateral resolution of the calculated PSF.

Axial FWHM

Displays the FWHM (Full Width Half Maximum) as a measure of the axial resolution of the PSF.

PSF View Section

This section shows you the PSF that is calculated for a channel based on the current settings. If you select the Auto Update checkbox, all changes made to the PSF parameters are applied immediately to the PSF view. This makes it possible to check quickly whether the settings made meet your expectations. You can extract the PSF from the image via right-click menu (PSF Snapshot) which opens the resulting new PSF document in the center screen area.

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
  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 .
  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 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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