ZEISS Lightsheet 7

Your Light Sheet Microscope for Multiview Imaging of Living and Cleared Specimens

 

Life sciences research can put big demands on your imaging capabilities: sometimes you need to image whole living model organisms, tissues and cells as they develop. Light sheet fluorescence microscopy (LSFM) with its unique illumination principle is ideal for fast and gentle imaging of such specimens. The exceptional stability of Lightsheet 7 lets you observe living samples over extended periods of time – even days – with less phototoxicity than ever before. What’s more, use this light sheet microscope to image very large optically cleared specimens in toto, and with subcellular resolution. Enhance your Lightsheet 7 with dedicated optics, sample chambers and sample holders to accurately adjust to the refractive index of your chosen clearing method, and then image your large samples, even whole mouse brains. All of this flexibility comes in this proven and stable boxed light sheet microscope from ZEISS.  

Highlights

  • Image Optically Cleared Specimens

Which optical clearing method you choose will depend on the type of tissue you are imaging, your fluorescent labels and the size of the sample itself. Lightsheet 7 is designed to match all of these different conditions. You can now image specimens at up to 2 cm in size at any refractive index between 1.33 and 1.58, and in almost all clearing solutions. This stable turnkey light sheet microscope lets you acquire overview images and data with subcellular resolution. Whether you work with optically cleared organoids, spheroids, organs, brains or other specimens, Lightsheet 7 is your microscope of choice for fast, gentle LSFM imaging.  

 

C57 BL6J mouse perfused with PBS, CellTracker™ CM-DiI Dye, and 4% PFA. Cleared using iDISCO+ protocol, final RIMS is Ethyl Cinnamate.

Sample Courtesy of:
Erin Diel – Harvard University; Harvard Center for Biological Imaging Room 2052, 16 Divinity Ave, Cambridge, MA 02138, USA

  • Get Best Image Quality and Stability

Take your LSFM imaging a step further to tackle a broad range of applications and achieve best image quality with your easy-to-use Lightsheet 7. Newly designed optics and sample chambers let you adjust to the perfect refractive index. The new sample holder makes mounting larger specimens simple. Smart software tools help you adjust imaging parameters, such as light sheet and sample positions, the right zoom settings, tiles and positions as well as data processing parameters. All of these new features go hand in hand with the reliable ZEISS combination of cylindrical lens optics and laser scanning to generate the illumination light sheet. Add the patented Pivot Scan technology and get artifact-free optical sections with best image quality.

Dedicated optics for your ZEISS Lightsheet 7
  • Observe Real Life – Fast and Sensitively

Your Lightsheet 7 now features the high quantum efficiency of pco.edge sCMOS detectors to enable observations of the fastest processes at the lowest illumination light levels. You'll get a real life view of your samples without the adverse effects of excitation light on their biology. For vertically oriented specimens and highest frame rates, opt for the CMOS detector Axiocam 702: a special sample chamber provides heating, cooling and CO2 to maintain the perfect environment for your experiments. Add Multiview and triggering options to control external devices – Lightsheet 7 is your ideal light sheet microscope to observe live processes in an almost unlimited range of organisms.

Development of Arabidopsis.
Arabidopis Flower Development – Sample: Courtesy of S. Valuchova, P. Mikulkova and K. Riha, Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.

The Principle of Light Sheet Fluorescence Microscopy

Light sheet fluorescence microscopy (LSFM) splits fluorescence excitation and detection into two separate light paths, with the axis of illumination perpendicular to the detection axis. That means you can illuminate a single thin section of the sample at one time, generating an inherent optical section by exciting only fluorescence from the in-focus plane. No pinhole or image processing is required. Light from the in-focus plane is collected on the pixels of a camera, rather than pixel by pixel as, for example, in confocal or other laser scanning microscopes. Parallelization of the image collection on a camera-based detector lets you collect images faster and with less excitation light than you would with many other microscope techniques. In summary, LSFM combines the optical sectioning effect with parallel image acquisition from the complete focal plane. This makes 3D imaging extremely fast and very light efficient.

The de-coupling of the detection optics from the illumination optics enables fluorescence excitation with dedicated lenses at low numerical aperture, without sacrificing detection resolution and sensitivity. This makes LSFM ideal for imaging of samples at the millimeter scale, such as developing organisms or large cleared tissue samples.

Lightsheet 7 LSFM illumination principle

How Lightsheet 7 works

 

Watch the animation to see how easy it is to position and image your samples with ZEISS Lightsheet 7.

The Patented Pivot Scanner

Delivers Homogeneous Illumination

When the light sheet is passing through the sample, some structures of the specimen, e.g. nuclei, will absorb or scatter the excitation light. This will cast shadows along the illumination axis, as you see in the left figure. This effect occurs in all fluorescence microscopes, but the illumination axis in light sheet fluorescence microscopy is perpendicular to the observation axis and so this effect is more obvious. In Lightsheet 7, a patented Pivot Scanner alters the angle of the light sheet upwards and downwards during image acquisition. By altering the illumination angle the shadows will be cast in different directions and excitation light will also reach regions behind opaque structures, as you see in right figure. This patented Pivot Scanner is a perfect way to acquire artifact-free images and to improve downstream processing and analysis steps. It is always better to tackle artifacts right at their origin.

ZEISS Lightsheet 7 at Work

Nephrology

Mouse kidney cleared with iDISCO protocol and imaged in ethyl cinnamate with ZEISS Lightsheet 7 detection optics 5× / 0.16 foc and Clr 20× / 1.0 nd = 1.53 (insert). The mouse was perfused with DyLight 594 conjugated tomato lectin to visualize vasculature and glomeruli (red). In green: auto-fluorescence to visualize tissue anatomy. 3D whole organ imaging and computational image analysis of glomerular size and number helps to gain a better understanding of the mechanisms of diverse kidney diseases, e.g. diabetic nephropathy. Processed with arivis Vision4D® on ACQUIFER HIVE.

 

Sample courtesy of U. Roostalu, Gubra, Denmark.

 

Sample courtesy of U. Roostalu, Gubra, Denmark.

Developmental Biology

3D Data set of a P10 mouse tr achea displaying the anatomical organization of mechanosensory nerve fibers. Staining: DAPI, Collagen IV (Alexa 488 antibody), sensorial fibers (reporter strain expressing tdTomato, Alexa 555 antibody), neurofilament protein NF200 (myelinated s nerve fibers, Alexa 647 antibody).
The sample was cleared in PEGASOS (Jing et al:, 2018, Cell Research) imaged in BB-PEG at RI of 1.54 with 5× / 0.16 foc detection optics and Cl r 20× / 1.0 nd = 1.53 respectively. 5× magnification data set: pixel scaling 0.61 × 0.61 × 1.63 micron, 3×3 tiles, Zoom 1.5×, 1230 z-sections, volume 2.57 × 2.58 × 2 mm 20× magnification data set: pixel scaling 0.23 μm × 0.23 × 0.58 micron, 1×5 tiles, Zoom 1.0×, 4206 z-sections, volume 2.0 × 0.45 × 1.82 mm.

 
 

Sample courtesy of P.-L. Ruffault, C. Birchmeier, Laboratory of Developmental Biology / Signal Transduction; A. Sporbert, M. Richter Advanced Light Microscopy; M. Delbrück, Center for Molecular Medicine, Berlin, Germany.

Vertebrate Limb, Spinal Cord Regeneration

Salamanders have the remarkable capability to regenerate their limbs and spinal cords. Molecular genetics tools allow to identify the stem cells responsible for this complex regeneration, and the injury-responsive signals that initiate their proliferation. This axolotl forearm has been cleared in ethyl cinnamate (Masselink, W. et al. Development 146, (2019)) and i maged with 5× / 0.16 foc detection optics at a refractive index of 1.57. The multi-tile data set was aligned, fused and rendered with ZEN imaging software and arivis Vision4D® software on an ACQUIFER HIVE data platform.

 

Sample courtesy of W. Masselink, Tanaka lab, Research Institute of Molecular Pathology, IMP.
Image courtesy of P. Pasierbek, K. Aumayr, IMP BioOptics, Vienna, Austria.

Neuronal Morphology

Imaging entire cells in the human brain is a task close to impossible, due to the tremendously sophisticated morphology of neurons- and their spread throughout the entire organ. Organoids allow the recapitulation of the human brain to a certain extent, including the production of neurons from neuronal stem cell cultures. With ECi clearing, neuronal morphology can be studied from the local to global level, which opens up fascinating possibilities for the study of neuronal morphology in 3D.
35 day old neuronal organoids sparsely labeled with GFP/tdtomato (3% GFP and 3% tdtomato) imaged with Clr 20× / 1.0 nd = 1.53 objective.
Pixel scaling: 222 × 222 × 567 nm.
Image volume: 1.66 × 0.66 × 1.6 mm.

 

Sample courtesy of D. Reumann and J. Knoblich, IMBA, Vienna, Austria.

Immunology

Imaging intact lymphoid organs in 3D allows to analyze and quantify the immune response to viral infection. T cells were transferred into wildtype host mice prior to harvest. The node was cleaned, fixed and cleared using Ce3D (Li et al. PNAS 163, 2017) prior to imaging at RI = 1.49 (ph 7) with a 5× / 0.16 detection optics (volume 2.5 × 2.5 × 1.6 mm). The image shows GFP labelled native CD8+ T cells (yellow), B cell follicles are stained using B220 (cyan) and the CD31 vasculature network (magenta).

Inguinal mouse Lymph node

Sample Courtesy of Joanna Groom, The Walter and Eliza Hall Institute of Medical Research, Australia

Mapping Vasculature of Entire Mouse Brain

A C57 BL6J mouse was perfused with PBS and 4% PFA. The brain was stained  perfusing Cell-Tracker™ CM-DiI Dye – a lipid dye to label the
vasculature membranes. The sample was cleared using iDISCO+ protocol,  equilibrated in ethyl cinnamate as final RIMS. It was then imaged in ethyl cinnamate at RI = 1.565 with detection optics Fluar 2.5× / 0.12 in a Translucence Mesoscale Imaging Chamber.

The high-resolution insert image on the right was acquired with Clr Plan-Neofluar 20× / 1.0 Corr nd = 1.53.
Image volume is 13.1 × 13.1 × 6 mm at a pixel resolution of 1.83 × 1.83 × 6.77 μm. It was acquired in about 40 minutes in 4×4 tiles, 866 z-sections.
Data volume is 93 GB. Data pr ocessed with ZEN imaging software and arivis Vision4D® on an ACQUIFER HIVE data platform.

 
 

Sample courtesy of E. Diel, D. Ric hardson, Harvard University, Cambridge, USA.

Mapping Interneurons and Purkinje Cells of Entire Mouse Brain

PV-tdtomato mouse brain was cleared using CLARITY protocol with final  imaging done in EasyIndex at a refractive index of RI = 1.46.
Parvalbumin-Cre yielding expression of tdtomato - Parvalbumin is expressed in a population of interneurons throughout the brain and in Purkinje cells in the cerebellum. The whole brain data set was acquired on ZEISS Lightsheet 7 with detection optics 5× / 0.16 foc. Image volume is 11 × 20 × 8.8 mm at a pixel resolution of 0.91 × 0.91 × 5.35 μm (12028 × 22149 × 1621 voxels). It was acquired in 6×10 tiles, 1621 z-sections. Data volume is 1.2 TB (805 GB after stitching). Data was processed with ZEN imaging software and arivis Vision4D® on an ACQUIFER HIVE data platform.

 

Sample courtesy of E. Diel, D. Ric hardson. Harvard University, Cambridge, USA.

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ZEISS Lightsheet 7

Light sheet fluorescence microscopy for Multiview imaging of living and cleared specimens.

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Technology Note: ZEISS Lightsheet 7

How to Get Best Images with Various Types of Immersion Media and Clearing Agents

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