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Lightfield 4D

This module provides instant volumetric imaging at high speed. You can acquire comprehensive 3D information with a single snap and avoid any time delay within an imaged volume. This allows you to capture the fastest movements within whole organisms at up to 80 volumes per second – with all spatiotemporal information intact. Crawling larvae, beating hearts, flowing blood, and firing neurons can be studied in 3D at high speed.

To best capture the essence of biological processes, imaging must be done in 4D, as both volume and time are essential for investigating living systems. The existing optical sectioning methods typically rely on sequential image acquisition to create z-stack images of volumes, which introduces time differences within the sample volume, severely limiting the imaging speed and the spatiotemporal accuracy of the acquired data.

Lightfield 4D offers a unique solution by imaging an entire volume at an exact point in time, without any time delay. Instead of capturing single 2D images at different time points, a micro lens array positioned in between objective and camera generates 37 individual images, collecting all the 3D information at the same instant. Each of these different views provides both spatial and angular information which serves as the foundation for creating a z-stack through deconvolution-based processing. This way, Lightfield 4D can generate 80 volume z-stacks per second.

In addition to the uniquely high speed of volume acquisition, this method is notably gentle on living samples. By utilizing a single illumination event for each generated volume, it eliminates the need for repeated illumination to capture individual image pixels or 2D images in order to acquire a sample volume, keeping light exposure short and to a minimum.

This combination makes Lightfield 4D the perfect method to capture fast processes as well as image data from multiple living samples over long periods of time. The resulting volume size depends on the selected objective lens. Its magnification and numerical aperture (NA) determine the area depicted and the reconstructed z-range. A variety of objective lenses are compatible with Lightfield 4D, achieving the ideal measurements for the desired sample volume and resolution.

The generated z-stacks are saved in the standard .czi file format, allowing for all the same rendering and analysis options as for any other z-stack created in ZEN. For reproducible, reliable, and trusted research, all 37 individual images are saved as raw data for your instant and future access.

Lightfield 4D Volume Color Coding

During a live acquisition, you can view the image data in the Volume Definition mode. The structures located at the top, center, and bottom of the current z-plane are displayed using a color-coded 3D maximum intensity projection of the processed image. The volume sections (Top, Center, Bottom) are treated as separate image channels. You can easily change the colors and toggle the visibility of these channels using the channel buttons in the Dimensions tab, see Dimensions Tab and Channels Section.

Setting Up a Lightfield 4D Track without Smart Setup

No Combination with other Tracks

Note that Lightfield 4D tracks do not work in combination with other track types, for example confocal tracks. If you have active Lightfield 4D tracks, other tracks are set inactive and vice versa.

Compatible Objectives

Lightfield 4D imaging is only available for certain compatible objectives. A message in the Experiment Manager section of the Acquisition tab (below Start Experiment) indicates if the currently selected objective is compatible with Lightfield 4D acquisition. In case it is not, use the Microscope tool in the Right Tool Area to change your objective.

  1. On the Acquisition tab, open the Imaging Setup tool, or the Channels tool.
  2. The track selection is displayed.
  3. Select Lightfield 4D.
  4. The dye selection dialog opens.
  5. Double click the dyes you want to use and close the dialog.
  6. The Lightfield 4D track is configured with an exposure time of 50 milliseconds.
  7. Time Series is activated and the Time Series tool opens.
  8. Acquire with High Speed is automatically activated if all channels are using the same filter position in the reflector changer. Channels requiring different filter sets cannot be combined in a high-speed acquisition. If you have defined two or more Lightfield 4D tracks for multi-channel experiments, the exposure time for each channel is set the same. Updating the exposure time of one channel updates it for all defined Lightfield 4D channels.
  1. You have set up a Lightfield 4D track. You can now configure the rest of your experiment and start it.
  2. Dimensions and functionalities that are not suitable for Lightfield 4D experiments (like Experiment Feedback) are deactivated after setting up the track.

Setting Up a Lightfield 4D Track with Smart Setup

Compatible Objectives

Lightfield 4D imaging is only available for certain compatible objectives. A message in the Experiment Manager section of the Acquisition tab (below Start Experiment) indicates if the currently selected objective is compatible with Lightfield 4D acquisition. In case it is not, use the Microscope tool in the Right Tool Area to change your objective.

Overwriting Settlings

All previously made channel and light path settings are overwritten by Smart Setup.

  1. On the Acquisition tab, click Smart Setup.
  2. The Smart Setup dialog opens. The LF4D mode is available if the correct hardware and license are available.
  3. Click LF4D.
  4. You are in Lightfield 4D setup mode.
  5. Click + Dye & Contrast Methods.
  6. The dye selection dialog opens.
  7. Double click the dyes you want to use and close the dialog.
  8. The dyes are added to the table and the Smart Setup displays Proposals for Specific Filter Settings.
  9. Select the proposal you want to use and click OK.
  10. The Smart Setup closes, a channel is created for every dye, and the light path is set up correctly.
  11. Time Series is activated and the Time Series tool opens.
  12. If you have selected the proposal for fastest acquisition, Acquire with High Speed is automatically activated if all channels are using the same filter position in the reflector changer. Channels requiring different filter sets cannot be combined in a high-speed acquisition. If you have defined two or more Lightfield 4D tracks for multi-channel experiments, the exposure time for each channel is identical. Updating the exposure time of one channel updates it for all defined Lightfield 4D channels in a high-speed acquisition.
  1. You have set up a Lightfield 4D track. You can now configure the rest of your experiment and start it.
  2. All dimensions and functionalities that are not suitable for Lightfield 4D experiments (like Experiment Feedback) are deactivated after setting up the track.

Setting Up and Starting a Lightfield 4D Experiment

Lightfield 4D Acquisition Always Uncompressed

Lightfield 4D images are always acquired uncompressed. To reduce the data size for Lightfield 4D images after acquisition, save them with Save As and select a compression.

Auto Save for Lightfield 4D Experiment

To set up Auto Save for your Lightfield 4D experiment, make sure to define the path in the Auto Save tool on the Acquisition tab. Set the path to a location on the SSD.

For high-speed acquisition, the images are saved in a temporary (tmp) folder in the Auto Save folder. After you save the fully acquired image, it is automatically removed from this tmp folder and stored in the selected location.

  1. You have set up one or more Lightfield 4D tracks, see Setting Up a Lightfield 4D Track without Smart Setup or Setting Up a Lightfield 4D Track with Smart Setup.
  1. To define the settings for your acquisition, use the tools on the Acquisition tab, for example by adapting the settings of the individual channels in the Channels tool.
  2. Your acquisition is set up.
  3. To define a time series experiment, Time Series has to be activated. It is automatically activated when you have added a Lightfield 4D track.
  4. The Time Series tool is available in the Multidimensional Acquisition section.
  5. Acquire with High Speed is automatically activated for all channels if all channels are using the same filter position in the reflector changer. Channels requiring different filter sets cannot be combined in a high-speed acquisition. If you have defined two or more Lightfield 4D tracks for multi-channel experiments, the exposure time for each channel is set the same. Updating the exposure time of one channel updates it for all defined Lightfield 4D channels. In this mode, the images from the camera are directly streamed to the hard drive (SSD) of the computer for maximal acquisition speed. It is not possible to set an interval.
  6. To define the duration and interval of your time series, open the Time Series tool.
  7. The time series is set up.
  8. To define positions for your experiment, see Defining a Lightfield 4D Multi-Position Experiment with the Tiles Advanced Setup or Defining a Lightfield 4D Multi-Position Experiment without the Tiles Advanced Setup.
  9. You have set up positions.
  10. Click Start Experiment.
  11. Your defined experiment starts, the image is displayed in the 2D view and the Lightfield 4D tab is displayed in the view options. The displayed image is the raw data image of the central lens.
  12. In case of high-speed acquisition, the update of the displayed image can contain a delay, as writing the data to the hard drive has a higher priority.
  13. In case you use Definite Focus and have configured your experiment to acquire with high speed, the Definite Focus is only executed before the time series starts and never during the high-speed acquisition.
  14. To display a specific time point during the experiment, go to the Dimensions tab and use the Time slider.
  15. The selected time point is displayed and the view is not updated based on experiment progress, Follow Acquisition is deactivated.
  16. To go back to an automated update of the image view, go to the Dimensions tab and reactivate Follow Acquisition.
  1. You have set up and started a Lightfield 4D experiment. Once the experiment is finished, the final image is displayed in ZEN.

Defining a Lightfield 4D Multi-Position Experiment with the Tiles Advanced Setup

  1. You have set up one or more Lightfield 4D tracks, see Setting Up a Lightfield 4D Track without Smart Setup or Setting Up a Lightfield 4D Track with Smart Setup.
  1. On the Acquisition tab, activate Tiles.
  2. The Tiles tool is available in the Multidimensional Acquisition section.
  3. Open the Tiles tool and click Show Viewer.
  4. The Tiles Advanced Setup view opens, see Tiles Advanced Setup. Note that the functionality of the advanced setup for Lightfield 4D experiments is limited to position experiments.
  5. To check the sample at the current stage position, click Live to start a live acquisition.
  6. In case of a live acquisition in the Tiles Advanced Setup, the live image opens as a separate image container and is also displayed at the stage position in the setup (blue rectangle).
  7. The Lightfield 4D tab with the view options for the acquisition is only available if you select the Live image container. To display view option tabs for both containers, go to the document bar on the right of the image containers, click and select Separate View Controls.
  8. Click Stop.
  9. The live image acquisition stops.
  10. Use the left and top toolbar of the view to define your positions. For detailed information, see Tiles & Positions with Advanced Setup.
  11. You have set up your positions. They are displayed in the view as well as the table of the Tiles tool.
  12. To change z-values of your positions or verify them, see Adjusting Z-Values of Positions.
  13. The z-values are changed or verified.
  14. Start your experiment by clicking Start Experiment, or continue setting up other parameters for your Lightfield 4D experiment, see also Setting Up and Starting a Lightfield 4D Experiment.
  15. You have defined multiple positions for your experiment.

Processing a Lightfield 4D Image with the Processing Function

  1. You have a Lightfield 4D image open in ZEN.
  1. Open the Processing tab and select the method Lightfield 4D Processing.
  2. The parameters for processing are displayed.
  3. Open the Input tool and select the image which should be processed.
  4. The image is set as input.
  5. Adapt the processing parameters, see Lightfield 4D Processing.
  6. The parameters are set up for processing.
  7. To define the location for your output file, go to the parameter Storage Location and click .
  8. A file browser opens.
  9. Select the folder where the processed image should be saved.
  10. You have defined the output folder. Note that this folder also defines the storage location for images created with the Lightfield 4D tab, see Processing a Lightfield 4D Image with the View Options Tab.
  11. To set the parameters individually for each channel of an image with multiple channels, activate Adjust per Channel.
  12. The Parameters tool displays all channels as a list. The currently selected channel is highlighted. For each channel you can use a dropdown to select whether to process it or to skip it, which means the channel is not processed and not part of the output image.
  13. Select the channels you want to process individually and set the parameters accordingly.
  14. You have set the parameters for each channel.
  15. On the Processing tab, click Apply.
  16. The image processing starts and the image is saved at the specified location.
  17. The processed image is opened.

Processing a Lightfield 4D Image with the View Options Tab

Storage location

You have to define or can change the storage location on the Processing tab, in the Lightfield 4D Processing function, see Lightfield 4D Processing and Processing a Lightfield 4D Image with the Processing Function.

  1. You have a Lightfield 4D image open.
  1. Go to the Lightfield 4D view options tab below the image and click Lightfield 4D.
  2. The parameters for processing are displayed.
  3. Adapt the processing parameters, see Lightfield 4D Tab.
  4. The parameters are set up for processing.
  5. To save your current parameter configuration, click Store.
  6. Your current parameter configuration is saved.
  7. Select which dimension of the image should be processed, for example Current Timepoint, and click Create Processed Image.
  8. The image is processed and saved at the location you have defined on the Processing tab.
  9. The processed image is opened.

Using Lightfield 4D in Direct Processing

Processing Not Possible on Acquisition Computer

For Lightfield 4D in Direct Processing, you cannot process the image on the acquisition computer. You must connect your acquisition computer to a dedicated processing computer running ZEN desk, see Connecting Acquisition Computer and Processing Computer

Lightfield 4D can be used in Direct Processing. For general information, see Direct Processing.

  1. 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.
  2. On the Acquisition tab, Direct Processing is activated. This activates the Auto Save tool as well.
  3. 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.
  4. If you want to execute an experiment with high-speed acquisition, the path for Auto Save has to be a location on the SSD of the acquisition computer! At the same time, the path for the output folder of Direct Processing should be set to a location on the processing computer (see steps further below). Always use folders where both computers have access to.
  5. On the Acquisition tab, you have set up your experiment for Lightfield 4D acquisition, see also Setting Up and Starting a Lightfield 4D Experiment.
  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 Lightfield 4D.
  4. The parameters are displayed, see Lightfield 4D Processing.
  5. To adjust the parameters individually for each channel of a multi-channel image, activate Adjust per Channel.
  6. The tool displays all channels as a list and the currently selected channel is highlighted. For each channel you can use a dropdown to select whether to process it or to skip it (the channel is not processed and not part of the output image).
  7. Define the settings for the parameters.
  8. The parameters are set.
  9. If you set up an experiment with high speed, open the Output section and deactivate Use Output Folder from Auto-Save.
  10. The location setting gets editable.
  11. Click .
  12. A file browser opens.
  13. Select a folder on the processing computer and click Select Folder.
  14. The new output folder is set.
  15. 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.
  16. 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.
  17. 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.
  18. 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.

Lightfield 4D Processing

This method enables you to process Lightfield 4D data. The Lightfield 4D reconstruction algorithm consists of two general steps, a rolling ball background subtraction and Richardson-Lucy deconvolution with total variation regularization.

Parameter

Description

Storage Location

Displays the location where the processed file is saved. Click to open a file explorer to change the location. Note that also processed images created with the Lightfield 4D tab below the image are saved at this location.

Adjust per Channel

Only available for images with multiple channels.
Activated: Opens a list with the channels to allow an individual adjustment of the parameters for each channel. For every channel you 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.

Iterations

Sets the number of iterations of a (constrained iterative) deconvolution. A higher value results in sharper objects but will also amplify noise and measurement errors. The available value range is between 5 and 50 iterations.

Background Correction

Sets the rolling ball radius to remove homogeneous background for better reconstruction. Larger values remove bigger structures, while smaller values also remove smaller structures and have a stronger effect on the image. The available value range is between 75 and 5 pixels.

Total Variation Denoising

Reduces negative effects of noise on the deconvolution by smoothing homogenous areas while preserving edges (sharp contrasts). The available value range is between 0 and 100%.

Lightfield 4D Tab

This view options tab is only visible if you have opened a Lightfield 4D image document.

Parameter

Description

Raw Data

Displays the raw data image of the selected single lens.

Selected Lens

Only visible if Raw Data is selected.
Selects the respective microlens of the Lightfield 4D multi-lens array.

Volume Definition

Only visible during live acquisition.
Activates the volume definition mode with a color-coded display of different regions of the image.

Volume Color Coding

Only visible if Volume Definition is selected.
Displays the colors for different z-areas in the image to find the center of your sample volume, see Lightfield 4D Volume Color Coding.

–

Top

Displays the color for image areas that are above focus.

–

Center

Displays the color for image areas that are in focus.

–

Bottom

Displays the color for image areas that are below focus.

Lightfield 4D

Only visible for fully acquired Lightfield 4D images and not during a live acquisition.
Displays the image with Lightfield 4D processing applied and displays the respective parameters for processing below. The Lightfield 4D reconstruction algorithm consists of two steps, a rolling ball background subtraction and Richardson-Lucy deconvolution with total variation regularization.

Iterations

Sets the number of iterations of a (constrained iterative) deconvolution. A higher value results in sharper objects but will also amplify noise and measurement errors. The available value range is between 5 and 50 iterations.

Background Correction

Sets the rolling ball radius to remove homogeneous background for better reconstruction. Larger values remove bigger structures, while smaller values also remove smaller structures and have a stronger effect on the image. The available value range is between 75 and 5 pixels.

Total Variation Denoising

Reduces negative effects of noise on the deconvolution by smoothing homogenous areas while preserving edges (sharp contrasts). The available value range is between 0 and 100%.

Parameter Configuration

 

–

Store

Stores the current parameter values.

–

Recall

Recalls parameter values from storage, if available.

–

Reset

Resets all parameters to factory defaults.

Process Image

Selects the image dimensions, for example Current Timepoint, to create a processed image with the currently selected parameter values.

Create Processed Image

Creates an image processed with the current parameter values. The processing always takes the currently displayed image. If you have an image of a multi-position experiment and deactivate the Scene checkbox on the Dimensions tab, all positions are displayed in the Image View and processed.

The image is saved at the location set in the Lightfield 4D Processing function, see Lightfield 4D Processing. To change the path in this tab, you have to change the storage location in the processing function and execute the function once on the Processing tab.

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