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Direct Processing

The development of microscopic techniques makes the data structure ever more complicated. In many cases, the raw data is large, time consuming to process and not needed at the end of the experiment. This module is designed to simplify the workflow to improve usability and to parallelize the acquisition and processing steps to save time.

During image acquisition using an acquisition PC, Direct Processing enables the user to select processing functions which are executed on a processing PC. Several different functions are available and you can define a sequence of functions (in a so-called pipeline), which are then executed sequentially one after another. Direct Processing starts to process the smallest processable entity as soon as its acquisition has been completed. In the case of deconvolution, this is typically a z-stack for one channel.

Direct Processing allows an acquisition computer to communicate with a second PC (processing computer) connected via a network connection. The acquisition computer instructs the processing computer to process images as they are being acquired. Before you can use the module, you typically need to connect the two computers. As there are numerous ways, how computers can be set up to become networked, we can only give some general advice here. Contact your local IT administration for help on how to configure the computers in accordance to the local infrastructure.

One way is to connect the computer controlling the microscope system to a second computer via a direct ethernet connection. If Network Discovery is switched on, Windows 10 will directly support such a point to point connection. Create a shared folder which can be accessed from both computers. Since no other network traffic will use this connection, the whole bandwidth will be available for saving data directly from the acquisition computer to a shared folder on the processing computer. This is the most efficient way as the acquired data do not have to be copied off the acquisition computer after the acquisition has finished.
Alternatively, it is also possible, to use a processing computer which is already integrated into an existing network. Depending on the network type and the kind of experiments being done, the bandwidth might not be sufficient to directly stream data to the processing computer. In such cases, to not limit the throughput of the acquisition, it is advisable to let the acquisition computer acquire data to a local drive and instruct the processing computer where to look for the acquired data.

If no processing PC is available, it is also possible to configure the same acquisition PC for parallel processing steps, i.e. acquire and process images on the same workstation.

For a communication via network, it is possible to link both computers with a discovery proxy. The discovery proxy is a service where you can register all available processing computers and your acquisition computer can then ask this service for the list of these available PCs. You can also link the PCs without such a discovery service, for which you then need the IP address and/or network name of the processing computer. Note that such a use of a discovery proxy is primarily designed for configurations in which you have more than one processing PCs.

Schematic of the function of a discovery proxy
Schematic of the function of a discovery proxy

System Requirements

Software Requirements

If processing and acquisition run on different computers and a second acquisition computer is added to the network, then the ZEN version of the computers can be different. In this case, the “older” ZEN version will not be able to use the added functionalities of the more recent ZEN version on the processing machine. There is no “backward” compatibility implemented.

Setting Up Your PC as Discovery Proxy

If you want to set up and use your computer as discovery proxy, take the following steps:

  1. Open Tools > Options > Direct Processing.
  2. Open the Setup Discovery Proxy Server section.
  3. The options for setting up the discovery proxy open.
  4. If your PC has several active network connections, under IP Address (this PC), select the IP address you want to use for the discovery proxy setup in the dropdown.
  5. Click Start.
  6. Click OK to close the Tools > Options dialog.
  1. This computer is now set up and used as discovery proxy with the displayed IP address.

Defining Settings in the Auto Save Tool

Auto Save and ZEN Connect

If you have opened a ZEN Connect Project, the folder in the Auto Save tool is automatically set to the folder where the ZEN Connect project is saved. In this case you cannot change the folder for Auto Save. Also note that with an active ZEN Connect project you cannot select the upload to ZEN Data Storage.

Using Direct Processing and ZEN Data Storage

If you are using the ZEN Data Storage server as the location for storing your data, all acquisition and processing computers involved must have access to the transfer share defined in ZEN Data Storage.

Also note that the images are only uploaded to ZEN Data Storage after they are now longer open in ZEN.

When using the Direct Processing functionality, perform the following steps on the acquisition computer to define the settings in the Auto Save tool.

  1. You have the Auto Save tool open on the Acquisition tab.
  1. In the dropdown, select if you want to save the image(s) locally or upload them to ZEN Data Storage.
  2. In the Folder field, specify the local directory, where acquired images should be stored. Make sure that it is a shared folder which can be accessed from both computers.
  3. If you select Store in ZEN Data Storage Server, make sure that the processing computer is also connected to the same server. Optionally, you can define if the image should directly be shared with a particular collection.
  4. If you want images to automatically be stored in a new subfolder named with the current date, activate Automatic Sub-Folder.
  5. In the Name field, specify the file base name for the acquired images.
  6. Activate Close CZI Image After Acquisition if you want to release the image from the acquisition computers memory.

Using Direct Processing with Airyscan Processing

  1. This function is only available if an Airyscan detector is available.
  2. If you are using Direct Processing on different computers, you have connected acquisition and processing computer, see Connecting Acquisition Computer and Processing Computer.
  3. 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.
  4. On the Acquisition tab, you have set up your experiment for image acquisition.
  5. On the Acquisition tab, Direct Processing is activated. This activates the Auto Save tool as well.
  6. 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.
  1. On the Acquisition tab, open the Direct Processing tool.
  2. If no Direct Processing settings were made before for the current experiment, a particular processing function is already preselected depending on your microscope, channel settings and licenses.
  3. In the Processing Function drop-down list, select Airyscan Processing.

    Note: If you uncheck the Auto Filter checkbox and activate Adjust per Channel, you can set the Super Resolution parameter in a channel specific manner.
  4. Select the desired settings for Airyscan Processing. For details how to use this function, see Airyscan Processing. Ideally, you have already checked the best parameters beforehand, using a sample image acquired under the same conditions as set up for the experiment.
  5. 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.
  6. 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 Airyscan processing will not take place.
  7. The processing computer reads incoming files and starts the Airyscan 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.
  8. To cancel the processing, 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 images. This should be done on the processing computer so that you immediately can 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 on the Direct Processing Tool on Acquisition Tab.
  2. When you open the image in the Image View, information about the executed Airyscan processing is available on the Info view tab. Additionally, general information about Direct Processing (e.g. the duration) is also available on the Info view tab of the processed image.

Direct Processing Tool on Acquisition Tab

Parameter

Description

Add Function

Adds another processing function to the pipeline.

Remove Function

Removes the currently selected processing function from the pipeline.

Pipeline

Displays a list of containers for possible processing functions. To set the function for the currently selected container, use the Processing Function dropdown list.
The functions in this pipeline are executed one after the other in the sequence as given here. You can change the position of a function in the pipeline via drag & drop.

Processing Function

Selects the processing function you want to use for Direct Processing for the currently selected container of the pipeline. Depending on your experiment, microscope, channel settings and licenses, a particular processing function is already preselected by default.

Parameter Section

In this section you have different parameters, depending on the selected Processing Function. For more information, see:

Adjust per Channel

Only visible for some functions and only if your experiment contains more than one channel.

Activated: Opens a list with the channels to allow an adjustment of each parameter for each individual channel. For every channel you also have the following options:

–

Process Channel

Processes this channel according to the set parameters.

–

Skip Channel

Skips this channel when processing. This channel will not be in the output image.

–

Copy Channel

This channel is copied into the output image without processing.

Use Advanced Settings

Only visible if you have selected Deconvolution or Apotome Plus.
Activated: Enables the dropdown menu to load an advanced setting.

Load Setting created in the Deconvolution function

Only available if Use Advanced Settings is activated and Deconvolution is selected.
Selects advanced settings which were created in the image processing function Deconvolution (adjustable).
Note: Currently Direct Processing only supports settings configured in the Deconvolution tab of the function, some settings on the PSF tab such as spherical aberrations correction cannot be used in Direct Processing.

Load Setting created in the Apotome Plus function

Only available if Use Advanced Settings is activated and Apotome Plus is selected.
Selects advanced settings which were created in the image processing function Apotome Plus (adjustable).
Note: Currently Direct Processing only supports settings configured in the Apotome Plus tab of the function, some settings on the PSF tab cannot be used in Direct Processing.

Output

Only visible if Show All is activated.
Displays options to adjust setting for the output image(s).

-

Output Folder

Displays the path where the processed image is saved. Make sure that a shared folder is selected where both the acquisition and the processing computers have access to.

-

Use Output Folder from Auto-Save

Activated: Synchronizes the output folder for Direct Processing with the folder in the Auto Save tool. The Output Folder can only be changed by editing the Folder in the Auto Save tool respectively. If you have opened a ZEN Connect Project, the folder in the Auto Save tool is automatically set to the folder where the ZEN Connect project is saved.

-

Create Sub-folders Automatically

Activated: Creates sub-folders in the output folder. All processed images end up in a new folder named with the current date.

-

Define File Naming

Defines the filename of the processed image. Default: processed.czi.

-

Use Original Name with Suffix

Activated: Uses the name of the file as it was acquired and adds the defined file naming as a suffix.

-

File Name Preview

Displays a preview of the name of the processed acquisition image.

-

Remove Acquired Image after Processing

Activated: Keeps only the processed image and deletes the acquisition image after processing.
Deactivated: Keeps both the acquired and processed image.

Handling of Processed Image

Only visible if Show All is activated.
Selects if/how the processed image should be displayed here on the acquisition PC.

-

Do Not Open Automatically

The processed image is not opened automatically.

-

Display in Split View

The processed image is displayed in a splitter document together with the acquired image as soon as the first image data is processed.

-

Only Open Final Image

The processed image is displayed after processing is completed.

Process On

Only visible if Show All is activated.
Selects where the processing of the image takes place.

-

This PC

Selects the option to process the data on this PC, i.e. the PC used for the acquisition.

-

Remote PC

Selects the option to connect to a separate (remote) processing PC for processing your data and displays an additional parameter to configure the connection.

Connected Processing PC

Only visible if Remote PC is selected.
Displays the status of your connection to a processing computer, for example to which PC you are connected and what the progress of your job is. If you are not connected to a PC, you can edit your connections here.

-

Edit Connection

Opens the dialog which lists all the connected PCs that are ready to receive remote processing requests.

Airyscan Processing Parameters

This set of parameters is only visible if Airyscan Processing is selected as Processing Function. For the information, you can also see Airyscan Processing.

Parameter

Description

3D Processing

Only available for images with 5 or more z-positions.
If activated, this option improves the resolution in axial and lateral direction. The data set needs to have at least 5 z-sections acquired with an optimal step size. 3D Processing is slower than 2D Processing. For 3D Processing, the whole z-stack (single channel and time point) needs to fit into the physical memory.

2D SR Processing

Only available for 2D images.
Enhances the 2D resolution. Note that this only results in increased super resolution when images are acquired with optimal settings and sufficient signal.

Auto Filter

If activated, a suitable Super Resolution parameter for the Airyscan processing is automatically determined for the selected data set. To manually adjust the Super Resolution parameter, deactivate the checkbox.

Strength

Use this option for an increased (high) or decreased (low) strength of the automatically assigned filter value. This is especially useful for 3D processing, as the 2D preview of the processing filter value in the Airyscan viewer does not allow to conclude the result after a 3D data processing.
The increment of this parameter is ± 0.4 compared to the standard auto Airyscan processing. This setting is not available when manual processing strength is selected.

Adjust per Channel

Only visible if Auto Filter is deactivated and only for images with two or more Airyscan channels.
If activated, you can manually set channel specific Airyscan processing parameters.

Super Resolution

Only available if Auto Filter is deactivated.

Manually adjusts the Super Resolution parameter.
Note: High strength might look attractive at some images, Z planes or color channels, but other filtering artefacts might occur which appear like small rings in the image. Also the results will become very sharp, but grainy. So carefully check your image data in order to avoid such artefacts.

Deconvolution Parameters

This set of parameters is only visible if Deconvolution is selected as Processing Function.

Parameter

Description

-

Simple, very fast (Nearest Neighbor)

Only available if Use advanced settings is deactivated. The Simple, very fast method is only available if you have configured an experiment with a z-stack acquisition.

In Direct Processing, the parameters for Deconvolution are based on Deconvolution (adjustable). The Normalization is set to Clip (with a Factor of 1) in case of remote processing because only with Clip the output images contain brightness values which allow quantitative comparisons.

For Direct Processing, you cannot change any other values.

We recommend Excellent, slow (Constraint Iterative). Note that this method is only available if you have the license for the Deconvolution module.

-

Better, fast (Regularized Inverse Filter)

-

Good, medium speed (Fast Iterative)

-

Excellent, slow (Constraint Iterative)

Direct Processing PCs Dialog

Parameter

Description

Use Discovery Proxy

Activated: Uses a Discovery Proxy server for the communication between the computers. This control is synchronized with the respective options in the Tools > Options > Direct Processing dialog.

–

Host Name

Only available if Use Discovery Proxy is activated. Displays and edits the name/IP address of the discovery proxy.

–

OK

Uses the defined Discovery Proxy.

Automatically Select Processing PC

Activated: Automatically selects a processing PC for each experiment based on the available PCs and their queue length (the PC with the shortest queue is selected). After the installation of ZEN, this option is activated automatically and remains so until it is deactivated.

Note that an automatic selection is not possible if you want to use the function Intellesis Denoising in Direct Processing.

Available Processing PCs

Displays a list with all the available processing PCs. It provides the name, an overview of how many jobs are currently in the Queue.
Your current PC is automatically listed if you have clicked Start Receiving in the Direct Processing tool on the Applications tab.

–

Connect

Connects to the respective processing PC.

–


Delete

Only available for PCs that are added as Custom Processing PC.
Deletes the custom PC from the list.

Custom Processing PC

Defines a custom PC for processing.

–

Host Name

Sets the name/IP address of the respective processing PC.

–

Port

Sets the port of the respective processing PC.

–

Add

Adds the defined custom PC to the list of available PCs.

Refresh

Refreshes the list of available PCs.

Close

Closes this dialog.

Direct Processing Tool on Applications Tab

Receiving by Default

By default, the computer is starting the receiving of processing requests automatically on application startup, i.e. Start Receiving is already active. If necessary, this behavior can be changed under Tools > Options > Direct Processing > Setup Processing PC.

Parameter

Description

Start/Stop Receiving

Starts or stops the reception of processing requests. The processing computer waits for processing requests from the acquisition computer. The computer is receiving by default.
Note: If you click Stop Receiving while an experiment is running, the experiment is continued and processed. Only after finishing the currently running experiment, a new experiment is processed.

Listening on Communication Path

Shows the path where the computer is listening for processing requests.

Current Request

Displays which image is currently being processed. A progress bar indicates how close you are to completing the currently processed image.

Items in the Queue

Displays the number of images to be processed. Note that due to the integrative nature of the CZI images, individual scenes will not show up as individual steps in the queue. Only when separated CZI documents are being produced, will the queue show a count > 0.

Cancel Processing

Cancels the processing of the images in the output folder.

Sharing a Folder for Direct Processing

This step by step guide gives instructions on how to create a shared folder for the Direct Processing functionality in ZEN.

You need an imaging system with its workstation (called acquisition computer) and for processing you need a high-end workstation (called processing computer) with a P6000 card (M/P4000 or M6000 works too) running the newest NVIDIA driver. Both computers need the software version ZEN 3.0 or higher.

Computer connection

The processing computer must be connected to the acquisition computer via ethernet connection. It works best to have both workstations equipped with a 10 GB ethernet card. A 1GB Ethernet is fine as well, just make sure to not create very large images and deactivate Follow Acquisition in ZEN when acquiring larger images. For details on how to connect your computer in a network, ask your local IT department. For some basic information, see also Connecting Computers via Cable.

IP Addresses

This is not strictly necessary, but knowing the IP Addresses is the safest way to troubleshoot networking issues, so we recommend to check this in any case. It is easiest to network the computers if both have the same login credentials, e.g. Username = ZEISS, password = zeiss. The computers can only network with each other if a password is set for both logins. For a description of how to look up the IP address of your PC, see Looking Up the IP Address of Your Computer.

Also note down the computer names. You can find those when you right click This PC in the explorer and select Properties.

Creating a Shared Folder on the Processing Computer

You have to make your processing PC discoverable in your network and create a shared folder.

  1. You are on the processing computer.
  1. Click on Windows start menu > Settings > Network & Internet > Status > Network and Sharing Center.
  2. The Network and Sharing Center opens.
  3. Click Change advanced sharing settings.
  4. The Advanced sharing settings window opens.
  5. Turn on network discovery for your network.
  6. Click Save changes.
  7. Open the Windows Explorer and create a folder where both data and communication between both ZENs should happen, e.g. “D:\direct processing”.
  8. Right click on the folder and select Properties.
  9. Go to the Sharing tab.
  10. Click Advanced Sharing.
  11. The Advanced Sharing dialog opens.
  12. Activate Share this folder.
  13. Click Permissions.
  14. The Permissions dialog opens.
  15. To give access to specific groups or people, click Add.
  16. The Select Users, Computers, Service Accounts, or Groups dialog opens.
  17. If you want to change which accounts you can add, click Object Types to open a dialog to select different types.
  18. If you want to change the (network) location, click Locations to open a dialog to select the location.
  19. In the text field, enter the user or group name you want to give access to and click Check Names.
  20. If the name exists, it is verified. In case there is no (exact) match, or in case there are multiple matches, a dialog opens where you can refine your search or select the right user/group.
  21. Click OK.
  22. The Select Users, Computers, Service Accounts, or Groups dialog closes and the user/group is added to the list on the Permissions dialog.
  23. Select the user(s)/group(s) and activate the checkboxes for the permissions you want to give them to this folder.
  24. Click OK to confirm all changes in all dialogs.
  1. You have created a shared folder on the processing computer. You can now setup the access to the folder on the acquisition computer.

Accessing the Shared Folder from the Acquisition Computer

On the acquisition computer, you need to map the folder from the processing computer to a drive letter.
Note: You could also do this by directly accessing the folder via the IP address, however, it is better to use the Windows mapping function as this enables some caching mechanisms which make the connection more reliable.

  1. You are on the acquisition computer.
  1. In Windows Explorer, open the Network location.
  2. Select the processing computer. Here you might need the computer name you noted down before.
  3. Select the shared folder of the processing computer.
  4. On the top of the Windows explorer, click Easy access and select Map as drive from the dropdown menu.
  5. The Map Network Drive dialog opens.
  6. Select the respective Drive for the Folder.
  7. A login screen opens.
  8. Enter the credentials of the processing computer.
  9. Click OK.
  10. Click Finish.
  11. The folder is mapped to your acquisition PC. The result will look something like this:
  1. You are now ready to setup the connection of the two computers in ZEN. Refer to the respective chapter in the software manual or the help in the software, which is accessible by pressing the F1 key.

Looking Up the IP Address of Your Computer

  1. In the Windows search, enter cmd.
  2. Select Command Prompt.
  3. The Command Prompt window opens.
  4. Type in ipconfig/all and press Enter.
  1. You can now see information for the network configuration of your computer. There you can look up the IP address of your computer. You have to make sure to select the correct Ethernet port, as ZEISS workstations usually have at least two. If only one is connected, this is the one which shows an IP address.

Connecting Computers via Cable

We recommend connecting the PCs in a network by using i.e. a rooter. In case a rooter cannot be used and two PCs are connected via a cable, consider the following remarks:

  1. Establish a network by cable.
  2. By default, a network/connection is established.
  3. As a test, check the IPs of the computers and ping each other.
  4. In case this fails, check the ethernet adapter and enable DHCP. Also check firewall notifications/settings.
  5. Ports used by ZEN are open by default, but could be blocked by various reasons (IT, Windows, firewall).
  6. If a port is blocked. try to set another port in the ZEN options for Direct Processing (see also the help for ZEN).
  7. You can also test more than one ethernet adapter by pinging the PCs.

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