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!
Discover the Subcellular Dynamics of Life
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. Dynamic imaging of full sample volumes with 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. Fast laser switching allows for imaging using up to three colors practically simultaneously, with minimized color crosstalk.
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.
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.
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.
Product Features
Standard Sample Carriers Usage
- Slides
- 35 mm dishes
- Chamber slides
- Multi-well plates
Fast and Gentle Sample Location
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.
Automatic Sample Leveling
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
Automatic Alignment of All Optical Elements
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.
Doubled Temporal Resolution with Two Cameras
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.
Unattended Long-term Experiments
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
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.
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.
Developing Life at Early Stages
Oocytes
Live Mouse Oocytes Arrested in Metaphase II
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, C.elegans, Drosophila
Zebrafish Embryo: Trafficking mRNA Molecules
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 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.