ZEISS LSM Lightfield 4D
Instant volumetric high-speed imaging of living organisms
ZEISS LSM Lightfield 4D
Do not miss the moment when life reveals its secrets.
Lightfield 4D is instant volumetric high-speed imaging. Capture extensive 3D information at the touch of a button, with no time delay in the imaged volume. For the first time, observe the fastest movements in whole organisms at up to 80 volumes per second – with all spatial and temporal information. Live model organisms, beating hearts, flowing blood, and active neurons can be studied in 3D at unprecedented speed, revealing the secrets of life.
The unique one-volume-per-click acquisition minimizes light exposure and efficiently captures thousands of volumes over extended periods without damaging your sample. Your productivity increases to unimagined heights, as a single acquisition run captures multicolor images at multiple positions within or between whole organisms, organoids, or spheroids.
As an integral part of ZEISS LSM systems, Lightfield 4D enables you to efficiently combine its fast volumetric imaging with any other LSM acquisition method: photomanipulation, superresolution, spectral, and even molecular dynamics data can be integrated into any live imaging session.
One Click. One Volume.
Capture spatial signals and fast dynamics without compromise.
Minimal Light Exposure. Maximum Information.
Observe whole organisms for as long as you want without altering life processes.
Fast Acquisition. Higher Throughput.
Examine different positions or numerous samples with instant volumetric imaging.
One Imaging Platform. Endless Possibilities.
Design your experiments innovatively and combine high-speed volume imaging with all the possibilities of an LSM.
One Click. One Volume. High-Speed Capture of Physiological and Neural Processes in 3D
Abstract
Life is constantly in motion. Many neural and physiological processes occur at very high speeds. Their spatial and temporal dynamics are therefore often difficult to capture accurately. While previous technologies have become faster, acquisition time still increases with sample volume. Fast processes such as neural activity or heartbeats therefore require compromises, either in volumetric information or in frame rate. With Lightfield 4D, you no longer have to compromise, as it captures 80 volumes per second in 3D without time delay. This allows you to track neural activity in zebrafish brains, tissue movement in developing Drosophila embryos, and moving structures in C. elegans larvae. The unique one-volume-per-click imaging ensures that no crucial processes are missed or distorted. Finally, particle tracking in entire volumes at high temporal resolution is possible. Start your experiments in no time – on your confocal microscope and without changes to sample preparation.
Investigating Morphology and Heart Wall Motion in the Developing Zebrafish Heart
Cherry expression in cardiomyocytes, acquired with ZEISS LSM 990 Lightfield 4D and Plan-Apochromat 20×/0.8 Air. Volume size: 723 × 723 × 430 µm³, exposure time 12 ms over a total of 1.2 seconds. Sample courtesy of Stone Elworthy and Emily Noël, School of Biosciences, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility of the School of Biosciences at the University of Sheffield.
Analyzing the morphology and motion of the embryonic heart in 3D is a real challenge because the heart beats continuously. The data were acquired from a zebrafish larva embedded in agarose (3 days post-fertilization). With ZEISS Lightfield 4D, the heartbeat could be imaged at 80 volumes per second. The video shows 3 complete heartbeats in 1.2 seconds, with cardiomyocytes resolved temporally and spatially. This enables cell segmentation and tracking with ZEISS arivis Pro. It is clearly visible that the cardiomyocytes follow the exact same path with each heartbeat.
Investigating the Flow of Insect Blood Cells (Hemocytes) in Drosophila Hemolymph
GFP-expressing hemocytes, acquired with ZEISS LSM 990 Lightfield 4D and Plan-Apochromat 20×/0.8 Air. Volume size: 723 × 723 × 430 µm³, exposure time 12 ms over a total of 2.5 seconds. Sample courtesy of Iwan Robert Evans, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility of the School of Biosciences at the University of Sheffield.
Investigating the flow of hemocytes (insect blood cells) through the hemolymph in vivo was nearly impossible for researchers due to the rapid three-dimensional movement. ZEISS Lightfield 4D offers the unique opportunity to image a large volume fast enough to follow this process under physiological in vivo conditions. Thanks to the unparalleled imaging speed of 80 volumes per second, cells are reliably resolved both spatially and temporally. The acquired data enable subsequent segmentation and automated tracking with ZEISS arivis Pro.
Minimal Light Exposure. Maximum Information. Gentle Observation of Whole Organisms Over Extended Periods
Abstract
Capturing 3D information from live samples has always been a challenge, especially with large sample volumes. Optical sections require sequential image acquisition and assembly into a Z-stack. Each section involves light exposure that is not limited to the illumination plane and quickly adds up to a harmful amount within the volume. Lightfield 4D is different: a complete Z-stack is acquired with just one illumination, minimizing light exposure and phototoxic effects. Live samples can be imaged over extended periods with high temporal density. This combination of excellent 3D imaging speed and extreme gentleness allows you to track the sample multicolor over long periods without affecting the recorded life activity. You can observe developmental processes, cell migration, vesicle movements, and other changes in tissues and organisms that last for several hours or even days, yet achieve the necessary temporal resolution to understand the dynamics.
Observing the Formation of Adipose Tissue in a Developing Drosophila melanogaster Pupa
52-hour-old Drosophila melanogaster pupa with cDB::eGFP expression in adult fat body precursor cells using the OK6-Gal4;Elav-Gal80 driver, 15 hours of overnight imaging with 12 positions and 10 animals, 500 ms exposure time per volume every 2 minutes. Courtesy of Ignacio Manuel Fernández Guerrero, Cellular Analysis Facility, MVLS-Shared Research Facilities, University of Glasgow. Data acquired at the Cellular Analysis Facility, University of Glasgow.
Visualizing tissue and organ development in live animals provides better insights into the factors involved in their regulation and dysfunction. An example is the developing fat body, which forms during the pupal stage of Drosophila. With ZEISS Lightfield 4D, you can keep pace with cell movement and obtain robust data for 4D tracking. Acquisition speeds are high enough to image multiple animals at any given time. This allows you to capture more data and increase throughput while maintaining high image data quality. Last but not least, the illumination is so gentle that imaging can be done overnight without affecting the viability of the organisms or the fluorophore intensity.
Long-term imaging of sensitive processes: Zebrafish ear in developmental morphogenesis
Zebrafish embryo, time-lapse movie of the developing ear vesicle, 2-3 days post-fertilization, ear epithelium labeled with GFP-CAAX, cell nuclei with RFP. Volumes of 4 different zebrafish embryo ears were acquired every 2 minutes over a period of 16 hours. Courtesy of Tanya Whitfield, Sarah Baxendale, School of Biosciences, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility, University of Sheffield.
The morphogenesis of developing organs requires complex coordination of various regulators and genomic elements. Insights into the influence of these components are best obtained by screening animals with different genetic defects and observing dynamic organ patterning in real-time. Lightfield 4D enables the acquisition of light-sensitive processes with sufficient resolution to track the morphological patterning of epithelial cells. The one-volume-per-click imaging not only ensures that no developmental processes are overlooked or lost amidst the Z-stacks but also makes it possible to image multiple animals in batch mode. This allows you to capture all events and increase throughput in your experiments.
Fast Acquisition. Higher Throughput. Accelerated Information Capture from Large Samples Using Multiple Markers
Typically, acquisition time for large volumes is the critical factor limiting throughput in imaging. Acquiring a large volume with a single click accelerates your experiments many times over. The unparalleled speed with which Lightfield 4D captures multicolor volumes increases productivity in experiments in many ways: in each session, you can image and analyze more samples than ever before – immediately improving experiment statistics. Compare multiple different sample cohorts of wild-type and genetically modified phenotypes, or samples with different drug treatments. Instead of many hours, you only need minutes to capture the necessary data. This gives you more time for advanced analysis and investigation of your datasets.
Efficient Volume Imaging of Cleared Spheroids with Subsequent Cell Counting
Cleared spheroid of a co-culture of HCT-116-GFP cells (colon cancer)/NIH-3T3-RFP cells (fibroblasts) with stained cell nuclei (Hoechst). Acquired in an InSphero Akura plate. The dataset was segmented with arivis Pro. Sample courtesy of InSphero AG. Schlieren, Switzerland.
Organoids and spheroids can provide data that is more meaningful than data derived from classical 2D cell culture models. However, conventional acquisition methods such as confocal point scanning or the use of spinning disk systems are time-consuming for acquiring Z-stacks. The imaging speed with Lightfield 4D enables advanced screening applications where higher throughput is crucial, and faster screening of numerous spheroids under similar and different conditions, for example, in drug screening and treatment studies.
High-Speed Imaging of Cancer Organoids for Assessing Disturbances
Colorectal cancer organoids, actin cytoskeleton labeled with Phalloidin (magenta), cell nuclei labeled with DAPI (blue). Acquired with a 40x objective with 100 ms exposure time per fluorophore. Courtesy of Nikki R. Paul, Cancer Research UK Scotland Institute, Glasgow. Data acquired at the Cellular Analysis Facility, University of Glasgow.
Organoids are popular biological models for analyzing properties in cancer systems, such as response to drug treatments, extracellular environments, and immune cell interactions. Imaging these large 3D structures and screening extensive sample sets is particularly time-consuming. Lightfield 4D enables 3D image acquisition at a speed of several organoids per second. This drastically increases throughput in screening large quantities compared to conventional microscopy methods.
One Imaging Platform. Endless Possibilities. Innovative Experimental Setup Through High-Speed Volume Imaging in Combination with All LSM Capabilities
Abstract
Laser scanning microscopes (LSMs) have proven to be extremely versatile microscopy systems. They combine superresolution and spectral imaging with high-quality optical sections of large samples, allowing the integration of additional fluorescence data and molecular dynamics measurements. This remarkable flexibility, combined with the gentle, instant volume imaging of Lightfield 4D, elevates your experiments to the next level: monitor neural activity in 3D and high speed, complemented by superresolution structural details captured by Airyscan. Track macrophage movement in a wound healing experiment and enhance your investigation with high-resolution details of the wound area. Utilize the photomanipulation capabilities of the LSM in bleaching, photoactivation, photoconversion, or ablation experiments, followed by gentle volume imaging. All this on the same microscope within the same experiment, without having to move the sample even once.
The Thinking Zebrafish: Analyzing Neural Activity in Developing Organisms
Imaging calcium signaling as a proxy for neural activity is a widespread technique in numerous model systems. These signals occur rapidly (in milliseconds) and therefore require high temporal resolution. Furthermore, the brain consists of densely packed neurons and glial cells, making high spatial resolution difficult to achieve. Many imaging techniques cannot simultaneously achieve high spatial and high temporal resolution. Often, calcium signaling is only captured in a single plane or in a very small volume. However, you only gain insight into the functioning of neural circuits when you track neural activity in as many neurons as possible simultaneously. ZEISS Lightfield 4D enables the acquisition of significantly larger volumes at a more than sufficient speed to track neural activity. This allows you to capture firing neurons that are 100 µm or even further apart, providing entirely new insights into neural circuits.
If specific neural morphology needs to be examined, high-resolution images of the areas of interest can be acquired with the same ZEISS LSM, either with confocal superresolution or Airyscan superresolution.
The video shows calcium signaling, a reporter for neural activity, in the zebrafish brain. The reporter intensity changes in the shortest possible time. Thanks to the large volume and high speed of Lightfield 4D, neurons that are more than 50 µm apart can be captured simultaneously.
Data acquired from a zebrafish larva (4 days post-fertilization) expressing the calcium reporter GCaMP6; imaged with ZEISS LSM 990 Lightfield 4D and LD C-Apochromat 40×/1.1 water immersion; image volume: 361 × 361 × 109 µm³; 10 volumes per second for a total of 1 minute (661 time points); exposure time 91 ms; intensity-coding LUT (low intensity blue, high intensity red to white).
Additional high-resolution data were acquired using the Airyscan-CO-BY mode.
Sample courtesy of Anton Nikolaev, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility of the School of Biosciences at the University of Sheffield.
Brightfield Microscopy from ZEISS Your Insight into the Technology Behind It
To faithfully capture the essence of biological processes, imaging must be done in 4D, as both volume and time are crucial when studying living systems. This concept itself is not new; numerous optical sectioning techniques have been developed over the past decades to meet precisely this requirement. However, these methods typically rely on sequential image acquisition, which is then assembled into Z-stack images. The resulting time differences immensely limit the imaging speed and the spatial and temporal accuracy of the acquired data.
Lightfield 4D offers a unique solution here, capturing a complete volume at a precise time point without any time delay. Instead of acquiring individual 2D images at different time points, a microlens array placed between the objective and the camera generates 37 individual images, capturing all 3D information at the same time. Each of these views provides both temporal and angular information, which form the basis for creating a Z-stack through deconvolution-based processing. This enables Lightfield 4D to generate up to 80 volume Z-stacks per second.
In addition to the uniquely high speed of volume acquisition, this method is particularly gentle on live samples. Each volume is generated with only a single exposure. The sample no longer needs to be illuminated multiple times to capture individual pixels or 2D images to form a sample volume. This minimizes and shortens light exposure as much as possible. This combination makes Lightfield 4D the ideal method for capturing fast processes over long periods – or for imaging data from multiple live samples.
The resulting volume size depends on the objective used. The magnification of the objective and its numerical aperture (NA) determine the imaged area and the reconstructed Z-range. You can choose from numerous objectives to achieve the optimal measurements for the desired sample volume and resolution for Lightfield 4D.
The generated Z-stacks are stored in the standard .czi file format for ZEN. This allows you to use the familiar rendering and analysis options as with all Z-stacks created in ZEN. As a basis for reproducible, reliable, and trustworthy research, all 37 individual images are stored as raw data for immediate and future access.
A microlens array placed between the objective and the camera generates 37 individual images, capturing all 3D information at the same time.
Each of the 37 views provides both temporal and angular information that contributes to the volumetric information of the sample. Lightfield 4D is capable of generating up to 80 of these volumes per second.
Z-stacks are generated using deconvolution-based processing and stored in the .czi file format. This allows you to use all rendering and analysis options in ZEN and arivis Pro.
Choose Your Platform
Brightfield Microscopy in Combination with LSM Flexibility
ZEISS LSM 910
Understanding the Fundamentals of Life
Compact confocal microscope for innovative imaging and smart analyses
ZEISS LSM 990
Freedom in Research
State-of-the-art multimodal imaging combined in a single confocal system
| Lightfield 4D (available for ZEISS LSM 910 and ZEISS LSM 990 on ZEISS Axio Observer) | |||||
| Magnification | 40× | 25× | 20× | 10× | |
| RI-Immersion | 1.333 | 1.333 | 1 | 1 | |
| Field of View | 20.4 mm | ||||
| Object Field Size | 361 × 361 µm² | 585 × 585 µm² | 720 × 720 µm² | 1,444 × 1,444 µm² | Deviation between systems up to 2% |
| Z-Stack Range | 109 µm | 278 µm | 430 µm | 1,712 µm | calculated |
| Acquisition Speed | up to 80 volumes per second | ||||
| Excitation Wavelength Range | 405–740 nm | ||||
| X/Y Resolution * | 2.2 µm | 3.5 µm | 4.4 µm | 8.8 µm | measured, deconvolved |
| Z Resolution * | 2.8 µm | 8.4 µm | 13.6 µm | 57 µm | measured, deconvolved with optimal number of iterations |
| Voxel Size XYZ | 0.7 × 0.7 × 0.9 µm³ | 1.12 × 1.12 × 2.7 µm³ | 1.4 × 1.4 × 4.4 µm² | 2.8 × 2.8 × 18 µm³ | |
| Stack Size XYZ * | 512 × 512 × 121 pixels³ | 512 × 512 × 103 pixels³ | 512 × 512 × 99 pixels³ | 512 × 512 × 95 pixels³ |
Recommended Objectives for Lightfield 4D
- C-Apochromat 40×/1.2 W Corr M27
- Plan-Apochromat 40×/1.3 Oil DIC M27
- LD LCI Plan-Apochromat 40×/1.2 DIC M27
- LD C-Apochromat 40×/1.1 W Corr
- LD LCI Plan-Apochromat 25×/0.8 Imm Corr DIC M27
- Plan-Apochromat 20×/0.8 M27
- EC Plan-Neofluar 20×/0.50 M27
- Plan-Apochromat 10×/0.45 M27
- Plan-Apochromat 10×/0.3 M27
- EC Plan-Neofluar 10×/0.3 M27
* Measured with beads in agarose (RI = 1.378) with air or water immersion and excitation/detection wavelength (marker) 488 nm / 525 nm (eGFP)
