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!

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.

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.}

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.}

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.

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:

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.

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

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.

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.

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 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

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.

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• 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).
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Cos7 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.
• 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.

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.

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.

• 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.
• 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.

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.