ZEISS Lattice Lightsheet 7

Long-term volumetric imaging of living cells

ZEISS Lattice Lightsheet 7 makes light sheet fluorescence microscopy available for live cell imaging at subcellular resolution – while also allowing you to use your standard sample carriers. With this automated, easy-to-use system, volumetric imaging of subcellular structures and dynamics over hours and days with best protection from photo damage becomes available to everyone. Discover the dynamics of life in unprecedented depth of detail – with the ease you never imagined possible!

ZEISS Lattice Lightsheet 7

Focus on Lattice Light-Sheet Microscopy: Past, Present and Future

WILEY Webinar | April 28, 2022

Join us for a WILEY webinar, focusing on the latest developments in lattice light-sheet microscopy. Hear about experiences of researchers using it for long-term volumetric imaging of living cells, first-hand reports how lattice light-sheet microscopes evolved, and the next generation ZEISS Lattice Lightsheet 7.

 

Discover the Subcellular Dynamics of Life

 
  • Amazingly Simple Access 
    Examine living specimens directly on your standard sample carriers
  • Next to no Photo Damage
    Watch the subcellular dynamics of life over hours and even days
  • Near-isotropic Resolution
    Reveal three-dimensional details in their true proportions
  • High-speed Volumetric Imaging
    Don’t miss an interesting event on your coverslip
  • Truly Simultaneous Two-Color Imaging
    Raise your experiments to new heights

Lattice Light Sheet Technology Made Accessible to Everyone

 

The importance of gentle light sheet imaging at high resolution cannot be overestimated for the study of subcellular processes. With Lattice Lightsheet 7, ZEISS makes access to the benefits of this advanced technology amazingly simple. Without having to adapt your usual sample preparation, you can examine living specimens directly on the standard sample carriers you already use for confocal microscopy. Complex alignment processes are performed automatically in this system so that you can focus your full attention on your experiments.

 
 

Next to no Phototoxicity and Bleaching

 

You want to watch the dynamics of life at subcellular resolution to study how the finest structures change over time. But your conventional imaging systems quickly reach their limits because they are too invasive and destroy what you are observing. Instead, ZEISS Lattice Lightsheet 7 provides lattice-structured light that automatically adapts to your sensitive samples, resulting in a massive reduction of photobleaching and phototoxicity, to allow your experiments to continue over hours and even days. The controlled incubation environment and an integrated auto-immersion mechanism enable unattended long-term experiments.

Video: LLC-PK1 cell undergoing mitosis. Cells are expressing H2B-mCherry (cyan) and α-Tubulin mEGFP (magenta), recording over a period of 25 hours.

High-speed Volumetric Imaging

 

The extremely fast image acquisition of ZEISS Lattice Lightsheet 7 enables up to three volume scans per second for each color channel. This high temporal resolution means no longer missing an interesting event on your coverslip. Near-isotropic resolution along the X, Y and Z axes gives you a three-dimensional image of your sample that reveals structural details in their true proportions. Two cameras and the specially designed excitation beam path allow for truly simultaneous imaging of two colors and quasi-simultaneous imaging of three colors.

Video: COS-7 cell transiently transfected with Tomm20-mEmerald and Calreticulin-tdTomato. The example shows ER wrapping around mitochondria and assisting mitochondrial fission.

 

The Technology Behind It

The Principle of Lattice Light Sheet Microscopy

Light sheet microscopy in general (also called Gaussian light sheet microscopy) is well known for its gentle imaging conditions at superior imaging speed. The groundbreaking concept of decoupling excitation and detection allows illumination of only the part of the specimen that is in the focal plane of the detection objective lens. By moving the sheet with respect to the sample and recording one image per focal plane, you can acquire volumetric data without exposing the out-of-focus sample areas.

Light Sheet Microscopy Principle
Conventional (Gaussian) light sheet microscopy splits fluorescence excitation and detection into two separate light paths, allowing to generate an inherent optical section by exciting only fluorescence from the in-focus plane.

Lattice light sheet microscopy combines the advantages of light sheet microscopy with near-isotropic resolution in the confocal range. Advanced beam shaping technology creates lattice-shaped light sheets that are significantly thinner than standard Gaussian light sheets and thus provide increased resolution at comparable imaging speeds. The lattice structure of the light sheet is created using a Spatial Light Modulator (SLM), then projected onto the sample after passing scanners that dither the lattice structure to create a smooth light sheet.

Lattice Light Sheet Microscopy Principle
Lattice light sheet microscopy overcomes the limitations of Gaussian beams (limited optical sectioning, limited field of view) and Bessel beams (strong rings, excitation of out-of-focus fluorescence) by generating long and thin light sheets to achieve subcellular resolution.

The ZEISS Implementation of Lattice Light Sheet Microscopy

During the development of Lattice Lightsheet 7, ZEISS gave special attention to user-friendliness and compatibility with conventional sample preparation techniques. An inverse configuration is the most important prerequisite to allow the use of standard sample carriers for high-resolution microscopy. The challenges resulting from an inverse configuration are mainly refractive index mismatches as fluorescence is emitted from the sample, passes through aqueous cell culture media, a tilted glass coverslip and water immersion, then into the detection objective.

Unrivaled ZEISS Optics

Special ZEISS proprietary optical elements in the detection beam path compensate for refractive index mismatches and enable you to image samples as easily and quickly as with a confocal microscope.

ZEISS Lattice Lightsheet 7 Optics
Schematic of sample carrier and core optics module with excitation objective (1), meniscus lens (2) and detection objective with free-form optics (3). Examples show imaging without (A) and with correction of refractive index changes (B).

Product Features

ZEISS Lattice Lightsheet 7 - Incubation Chamber
Incubation chamber loaded with a standard 35 mm dish.

Standard Sample Carriers Usage

ZEISS Lattice Lightsheet 7 - Incubation Chamber
Incubation chamber loaded with a standard 35 mm dish.

Without having to adapt your usual sample preparation, you can examine living specimens directly on the sample carriers you already use for confocal microscopy. ZEISS Lattice Lightsheet 7 can be used with all standard sample carriers that come with a no. 1.5 coverslip for the bottom:

  • Slides
  • 35 mm dishes
  • Chamber slides
  • Multi-well plates
ZEISS Lattice Lightsheet 7 - LED Illumination
Gentle transmission illumination ensures that your sample is quickly located.

Fast and Gentle Sample Location

ZEISS Lattice Lightsheet 7 - LED Illumination
Gentle transmission illumination ensures that your sample is quickly located.

With the integrated transmission LEDs and oblique detection which provide a DIC-like contrast, you can easily locate your sample. Change from white to red transmission LEDs for more gentle illumination if necessary. And you can choose to include transmitted light illumination during long-term observations.

ZEISS Lattice Lightsheet 7 - 5-axis Stage
The 5-axis stage combines highest precision and speed with a large travel range for multiwell plate imaging.

Automatic Sample Leveling

ZEISS Lattice Lightsheet 7 - 5-axis Stage
The 5-axis stage combines highest precision and speed with a large travel range for multiwell plate imaging.

Specifically designed for this system, the unique 5-axis stage not only allows movement along the X, Y and Z axes, but also tilting with the highest precision in X and Y, compensating for even the smallest deviations in carrier dimensions or sample position. Leveling your sample is done automatically, which relieves you of tedious manual procedures.

ZEISS Lattice Lightsheet 7 - Beam Path
Schematic of the ZEISS Lattice Lightsheet 7 beam path (click to enlarge).

Automatic Alignment of All Optical Elements

ZEISS Lattice Lightsheet 7 - Beampath
Schematic of the ZEISS Lattice Lightsheet 7 beam path (click to enlarge).

For the best imaging results, the lattice light sheet must be adapted to each sample; therefore, ZEISS has implemented automatic alignment of all optical elements to eliminate time-consuming manual adjustments. The innovative design of the excitation beam path allows for rapidly changing laser lines without having to reprogram the SLM. This enables virtually simultaneous acquisition of multi-channel data sets so that you will not miss any events occurring in your sample.

ZEISS Lattice Lightsheet 7 - Dual-Camera Configuration
Dual-camera configuration of ZEISS Lattice Lightsheet 7.

Doubled Temporal Resolution with Two Cameras

ZEISS Lattice Lightsheet 7 - Dual-Camera Configuration
Dual-camera configuration of ZEISS Lattice Lightsheet 7.

The innovative design of the excitation beam path allows simultaneous excitation of the sample with multiple laser lines. Combined with two Hamamatsu ORCA-Fusion cameras, this enables truly simultaneous imaging of two channels, which is critical for a range of applications such as ratiometric experiments. A dual-camera setup also allows you to use single bandpass filters in front of each camera to minimize crosstalk and achieve cleanest results without compromising speed.

ZEISS Lattice Lightsheet 7 - Autoimmersion
ZEISS Lattice Lightsheet 7 autoimmersion equipment.

Unattended Long-term Experiments

ZEISS Lattice Lightsheet 7 - Autoimmersion
ZEISS Lattice Lightsheet 7 autoimmersion equipment.

Incubation

An integrated incubation system provides long-term stability throughout varying environmental conditions. The microscope controls and monitors temperature, CO2 and O2 levels, and humidity automatically, to preserve the integrity of your sample throughout the experiments. The lid with glass window allows quick and easy access to the sample to facilitate its inspection during an experimental run.

Autoimmersion

Prime the system to release any air, then a supply of immersion media tailored to the needs of your experiments is released automatically. Replenishing the immersion media is software-controlled, so you don’t have to worry about interfering with image acquisition. The reservoir is protected from illumination to keep bacterial growth at bay. Objectives are shielded from immersion supply; hence they remain dry, even if excess immersion media is applied.


Typical Applications

Typical Application Typical Samples Tasks

Live cell imaging

  • Adherent cells
  • Suspension cells
  • Volumetric imaging of subcellular processes with high speed: organelle morphology and dynamics, organelle-organelle interactions, vesicle trafficking
  • Volumetric imaging of membrane dynamics
  • Volumetric imaging of immune cells such as T cell mobility and activation
  • Gentle imaging of live cells for hours up to days with minimal phototoxicity and photobleaching
  • Cell proliferation and apoptosis assays

3D cell culture

  • Spheroids
  • Organoids
  • Cysts
  • Cells in hydrogel
  • Live imaging of spheroids or organoids with diameters up to 200 μm
  • Organoid self-organization
  • Cell migration and proliferation within organoids
  • Imaging of cell-cell interactions, 3D organization, migration and morphology
  • In vitro imaging of neuronal activity

Small evolving organisms

  • Zebrafish embryos
  • C. elegans embryos
  • Drosophila embryos
  • Resolving structural detail in 3D with close to isotropic resolution
  • Fast imaging of cellular and subcellular dynamics in embryos and small organisms up to 100 μm in diameter
  • Cell migration, cell-cell interaction, cell cycle, vesicle trafficking

Oocytes

 

  • Live imaging of whole oocytes in 3D with subcellular detail

Expanded samples

 

  • Water based gel expanded small samples

ZEISS Lattice Lightsheet 7 at Work

Lamin B1 in Action

 

Lamin B1 localizes to the nuclear envelope and is involved in disassembling and reforming the nuclear envelope during mitosis. The formation of so-called ‘nuclear invaginations’ has been reported frequently for many different cell types during mitotic events at different stages of the cell cycle. Nuclear invaginations can manifest as tubular structures that extend from the nuclear envelope and cross through the nucleus. Although these unique structures have been reported frequently, most research so far has been done with fixed cells. Consequently, the function of these structures is largely unknown even though plenty of hypotheses have been proposed.

This data set was recorded with a cell line from the Allen Institute for Cell Science in Seattle: human induced pluripotent stem cells which endogenously express mEGFP-tagged lamin B1 (AICS-0013). The overnight experiment was recorded for close to 8 hours with one volume imaged every 1.5 min. Cells going through mitosis can be observed throughout the whole duration. Formation and dynamics of nuclear invaginations can clearly be observed i n most of the cells, throughout the complete cell cycle.

Gentle illumination is crucial for imaging mitosis as this process is extremely delicate and light sensitive. To prevent replication of damaged DNA, cells arrest mitosis as soon as there is any damage from excitation light. The gentleness of Lattice Lightsheet 7 imaging and an extremely stable system is required for imaging mitotic events over longer time periods. Fast volumetric imaging in combination with near-isotropic resolution allows for looking at the sample from every angle and investigating unique subcellular structures in every detail. ZEISS Lattice Lightsheet 7 is the perfect tool for challenging experiments like this. Applications that were impossible before turn into reality – and with its ease of use, they can also become real for your research.

Subcellular Dynamics at Highest Volume Speed

 

COS-7 cells transiently transfected with Calnexin-mEmerald and EB3-tdTomato. EB3 labels the growing ends of microtubules and is necessary for the regulation of microtubule dynamics. Calnexin is a protein of the ER where proteins are synthesized. One volume every 7 sec; continuous imaging for 24 mins. Imaged volume: 118 × 113 × 22 μm3. 240,600 images, 401 volume planes for 300 time points.

 

Time lapse movie showing dynamics of a U2OS cell stably expressing Actin-GFP (cytoskeleton, cyan). Cells were also labeled with MitoTracker™ Red CMXRos (Mitochondria, green) and Draq 5 (Nucleus, magenta).

 

U2OS cell expressing Lifeact-tdTomato undergoing mitosis during continuous imaging. Maximum intensity projection. The cell was imaged constantly for 2.5 hours; one volume (113 × 90 × 11 μm³) every 2.2 secs. A total of 1,404,000 images was recorded; 351 volume planes for 4,000 time points.

 

COS-7 cells transfected with ER-targeted StayGold fluorescence protein. Maximum intensity projection. Recorded with 1 ms exposure time for 40 min continuously. 802,000 images in total. One volume (105 × 56 × 14 µm³) per 1.1 seconds. Sample courtesy: Mayawaki Lab, University of Tokyo, Japan.

 
 

Cos 7 cells transiently transfected with mEmerald-Rab5a and Golgi7-tdTomato. Golgi7 is a protein associated to the Golgi and Golgi vesicles. Rab5a is an early endosome marker. Tracking of vesicles in 3D with near-isotropic resolution becomes reality. Tracking was performed in arivis Vision4D®.

 

U2OS cells expressing Lifeact-tdTomato and stained with MitoTracker Green. Top row: single-camera configuration. Bottom row: dual-camera configuration. Crosstalk is minimized. In addition, twice as many images can be acquired, resulting in a doubling of temporal resolution.

 

T cell expressing Lifeact-GFP. Color-coded depth projection and maximum intensity projection side- by-side. The T cell was imaged constantly for over 1 hr; one volume every 2.5 secs. Sample: courtesy of M. Fritzsche, University of Oxford, UK.

Reliable Investigation of Co-Localization

As you want to investigate co-localization, you can’t afford any crosstalk to be confident the observed co-localization is real. However, the choice of using single-bandpass filters means you need to switch filters while imaging and this slows down the acquisition enough to cause significant shift between structures that you know should overlay. So, you can’t be confident in the co-localization results and observed interactions. A dual-camera setup solves this dilemma, giving you confidence in the acquired data and the results you can draw from it.

 

U2OS cells stained with MitoTracker Green (green) and MitoTracker Red CMXRos (magenta), two dyes that localize to mitochondria and should therefore always co-localize. Left: single-camera setup. A recording time delay between the two channels manifests in a spatial shift of the structures. Right: dual- camera setup. The structures overlay completely as is to be expected. 60 time points were acquired while with a single camera, only 16 time points could be acquired within the same time.

Ratiometric Experiments

The fluorescence intensity ratio of MitoTracker Green and MitoTracker Red CMXRos was analyzed to investigate mitochondrial membrane potential as only the uptake of MitoTracker Red CMXRos is membrane potential dependent; MitoTracker Green is a measure for mitochondrial mass but independent of mitochondrial membrane potential and can serve as internal reference. Thus, the fluorescence ratio of the two dyes is a relative measure of the mitochondrial membrane potential.

 

U2OS cell stained with MitoTracker Green (green) and MitoTracker Red CMXRos (magenta).

 

Fluorescence intensity ratio of MitoTracker Green and MitoTracker Red CMXRos.

Developing Life at Early Stages | Oocytes

 

Fixed mouse germinal vesicle oocytes stained for the nuclear envelope (anti-lamin, cyan), actin (phalloidin, magenta), and microtubules (anti-tubulin, yellow). The Sinc3 15 × 650 lattice light sheet was used for high-resolution imaging of microtubule and actin structures. Follow the 3D structure of the microtubules in the movie. Sample: courtesy of C. So, MPI Göttingen, Germany.

 

Fixed mouse germinal vesicle oocytes stained for the nuclear envelope (anti-lamin, cyan), actin (phalloidin, magenta), and microtubules (anti-tubulin, yellow). The Sinc3 100 × 1,800 lattice light sheet was used for imaging of the whole oocyte. Sample: courtesy of C. So, MPI Göttingen, Germany.

 

Live mouse oocytes arrested in metaphase II and stained for mitochondria (cyan), microtubules (magenta) and chromosomes (yellow). Sample: courtesy of C. So, MPI Göttingen, Germany.

Developing Life of Small Evolving Organisms | Zebrafish

 

DeltaD-YFP transgenic zebrafish embryo (Liao et al. 2016, Nature Communications). Fusion protein driven by a transgene containing the endogenous regulatory regions, expression in the tailbud and pre-somitic mesoderm. Signal visible in the cell cortex, and in puncta corresponding to trafficking vesicles (green). Nuclei in magenta. The embryo was imaged for 5 minutes constantly; one volume (150 × 50 × 90 μm3) every 8 sec. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

 

High-speed movie of zebrafish embryo. Volumetric imaging of trafficking mRNA molecules (green). Nuclei are shown in magenta. Data is displayed as maximum intensity projection. One volume (86 × 80 × 12 μm3) was recorded every 2.5 sec. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

 

Trafficking mRNA molecules were tracked in arivis Vision4D®. The movement of the zebrafish embryo was first corrected using a nucleus reference track. Then individual mRNA molecules were tracked over time to result statistics such as speed and directionality. Sample: courtesy of Prof. Andrew Oates, EPFL, Switzerland.

Developing Life of Small Evolving Organisms | C. elegans Embryos

 

C. elegans embryo stained for nuclei. The movie shows a color-coded depth projection of the embryo. The embryo was imaged for 10+ minutes constantly; one volume every 700 msec. Imaged volume: 115 × 50 × 30 μm³. A total of 101.000 images was recorded; 101 volume planes for 1000 time points. Customer sample.

 

C. elegans embryo stained for nuclei. The movie shows a color-coded depth projection of the embryo. The embryo was imaged for 19+ hrs every 5 mins and can be observed going through its normal sleep-wake cycle. Imaged volume: 115 × 50 × 30 μm³. A total of 23,836 images was recorded; 101 volume planes for 236 time points. Customer sample.

 

C. elegans embryo at the late bean stage (~400 min post fertilization) with ~560 nuclei marked with HIS-58::mCherry (magenta) and centrioles marked by GFP::SAS-7 (green). Cells in mitosis show condensed signal of HIS-58::mCherry and centrioles at spindle poles. Sample: courtesy of N. Kalbfuss, Göncy Lab, EPFL, Switzerland.

Developing Life of Small Evolving Organisms | Drosophila Embryo

 
 

Drosophila melanogaster is a model organism in many research fields such as biomedical research. Many genetically modified variants are available to researchers. This video shows a drosophila embryo with GFP labeling as it moves over time. A total of 91,100 i mages were taken, 911 volume planes, 100 time points. One volume, every 15 secs; imaging duration 25 mins, imaging volume: 300 × 455 × 145 μm3.

Developing 3D Cell Models

 

Spheroids and organoids are in vitro models of organs – much smaller and simpler but easy to produce and thus for developmental biologists an invaluable tool to study organ development. Unlike cell cultures, which usually consist of a monolayer of cells only, cells in spheroids / organoids form three-dimensional structures, allowing for the investigation of cell migration and differentiation inside 3D cell models. With lattice light sheet microscopy, imaging the development and self-organization of organoids becomes reality. Here, we can see a 3D rendering of a spheroid consisting of cells expressing H2B-mCherry (cyan) and α-Tubulin-mEGFP (magenta). Not every cell is labelled.

Developing Plants and Plant Seeds | Pollen Grain & Pollen Tube

 

Watch mitochondrial dynamics inside the pollen tube. Mitochondria move towards the tip at the edges and back in the middle of the tube. While trafficking, mitochondria constantly fuse and divide for repair processes and to share and distribute biological molecules. Sample: courtesy of R. Whan, UNSW, Sydney, Australia.

 

Pollen tube stained for mitochondria (MitoTracker Green, green) and Lysosomes (Lysotracker Red, red). Watch the pollen tube extend from the crack in the pollen grain (visualized by its autofluorescence). Mitochondria don't quite advance to the very tip of the pollen tube but stop a few microns before the tip. Rendering of the data set was performed in arivis Vision4D®. Sample: courtesy of R. Whan, UNSW, Sydney, Australia.

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ZEISS Lattice Lightsheet 7

Long-term Volumetric Imaging of Living Cells

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