Cleared Tissue LightSheet

High-Speed High-Resolution Imaging of Cleared Tissue and Organs

Highlights

Features

Data Sets

Specifications

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High-Speed High-Resolution Imaging of Cleared Tissue and Organs

Cleared Tissue LightSheet (CTLS) is a dual-side illumination lightsheet microscope for whole organ imaging. It incorporates a fully automated macro zoom microscope with high NA objectives and a spatial light modulator (SLM) to optimize the lightsheet for high-resolution imaging. Using a back-thinned sCMOS camera, CTLS images entire organs at high-resolution with low photobleaching. A volume of one cubic centimeter can be imaged in less than an hour.

Slide

Back-thinned sCMOS Camera

5.3MP 30fps 95% QE scientific CMOS detector

Computers and Storage

Macro Fluorescence Objectives

LaserStack Laser Combiner

Computer workstations featuring Intel Xeon processors, NVIDIA Quadro GPUs and high-speed scalable SSD storage
Petabyte data storage systems ranging from 500TB to over 2PB

PlanApochromat 0.5x/0.125NA
PlanNeoFluar 1.0x/0.25NA
Apochromat 1.5x/0.37NA
PlanNeoFluar 2.3x/0.57NA

Modular Laser Combiner
Up to 8 lasers
Up to 300mW

Automated Macro Zoom Microscope

Spatial Light Modulator

Chambers and Specimen Holders

Motorized focus, zoom and filter selection

Dynamic lightsheet focus
Infinity Beam multi-directional illumination
Axial chromatic aberration correction

PTFE sample holders compatible with both aqueous and organic clearing solutions
Temperature controlled chamber
Multi-specimen holder for up to 7 different samples
or a whole cleared animal

Dual-Side Illumination

5x/0.14NA and 10x/0.28NA options

Powerful SlideBook Lightsheet Software

3i’s SlideBook software CTLS control module manages every step in cleared tissue imaging. An intuitive workflow guides users through the collection of 3D stacks, 3D data montaging, volume rendering and finally movie making with story-board support. SlideBook is GPU optimized and readily handles the creation and processing of 3D datasets at over 1TB, making them ready for analysis and rendering. SlideBook .SLD files can be accessed via any application supporting Bio-Formats OME, allowing seamless collaboration in any workflow.

Slide

User-Selectable App Appearance

Select a color scheme from dozens of options
Switch on-the-fly from dark to light themes

SlideBook Open File Format

Directory-based open file format for big data and high performance computing applications

Volume Rendering

3D and 4D volume view visualization tools support a user- specified bounding box and a storyboard interface where multiple perspectives can be assembled into a single movie

NVIDIA CUDA GPU Acceleration

GPU acceleration of computationally-intensive operations such as deconvolution

Montage

Montaging of 3D data is built into SlideBook’s workflow with spatial and frequency-based algorithms

Prescan Roadmap

The CTLS console is a single easy-to-use window featuring all frequent controls and status displays from laser selection to prescan roadmap to capture

CTLS includes motorized zoom lenses to automatically zoom out and create a 3D map of the entire specimen. This map serves as virtual eyepieces, allowing inspection of the entire specimen at higher magnification and identification of regions of interest for zoomed-in high-resolution 3D imaging

System Capture Console

Capabilities

Capture

Control hundreds of devices including microscopes, stages, lasers, wheels, piezos, scanners, shutters and much more.

View

Visualize data through any numbers of portals, from single images to z-stacks, time lapse, color channels and 4D views.

Analyze

Analyze images and extract statistical data via a wide variety of algorithms while maintaining original data integrity.

Scripting

Macro scripting for capture and analysis enhances the flexibility and power available to users.

Communicate

Present and export data easily as 16-bit TIFFs, 3D movies, graphs or spreadsheets. Data is directly portable to MATLAB and Excel and adheres to Open Microscopy Environment (OME) standards.

Partners

MATLAB

Through hierarchical and conditional capture, user-supplied MATLAB programs can control experimental workflows.

Microvolution

Microvolution software delivers nearly instantaneous deconvolution by combining intelligent software programming with the power of a GPU.

Aivia

Aivia is an innovative and complete 2D-to-5D image visualization, analysis and interpretation platform with artificial intelligence-guided image analysis.

Dell

The latest high-power computer workstations control all microscope hardware and enable high-speed processing, segmentation and volume rendering of terabyte (TB) datasets.

Highlights

Dynamic Lightsheet Focus

CTLS forms a dynamic lightsheet by sweeping a Gaussian beam with a high-speed galvanometer across a typical 2mm field of view. An SLM rapidly translates the thinnest part of the sheet along the axis of propagation. Translating the optimal center of the beam across the field results in thin optical-sections with superior axial resolution.

 

 
Above: A “tiled” Gaussian beam tuned to optimize axial resolution and optical sectioning across a 2mm field of view. Left: A bumblebee cleared with iDISCO imaged with 1.5x/0.37NA shown as a high resolution capture. The video demonstrates Cleared Tissue LightSheet’s flexibility from high speed to high resolution capture. Specimen courtesy of Dr. Ruth Hans, Light Microscopy Facility, Technische Universität Dresden, Center for Molecular and Cellular Bioengineering (CMCB) Technology Platform.

Cleared Tissue LightSheet XL

CTLS-XL incorporates a large chamber more than twice the size of standard chambers. The XL may be used to image an entire mouse or large organs roughly 20mm in width and 60mm in length. A 0.5x imaging objective with 114mm of working distance allows for exceptionally fast imaging of very large samples. All CTLS excitation and detection objectives and the smaller glass chambers are compatible with the XL microscope. CTLS-XL represents the ultimate toolkit for cleared tissue imaging of whole animals, large organs, or smaller specimens using chambers that require a modest amount of index matching media.

Shown below is a Thy1-YFP mouse measuring 4.5cm in length cleared with PEGASOS and imaged with Cleared Tissue LightSheet XL (sample Courtesy of Dr. H. Zhao, Beijing Institute for Brain Research). The ability to image whole, cleared animals is a useful investigatory tool in many areas of research including neuroscience, tumor biology, and more.

Features for Advanced Cleared Tissue Imaging

Dual-Side Illumination

A pair of excitation objectives are mounted in 3-axis focus mounts for easy beam centration and alignment. Illumination is toggled from the left side to right and back by the high-speed galvo. This allows for optimal sheet penetration across wide specimens with even illumination and neutralization of shadows caused by opaque structures.

Left Side Illumination

Mouse brain illuminated from the left side

Right Side Illumination

Mouse brain illuminated from the right side

Dual-Side Illumination

Mouse brain illuminated from both sides
Illumination Objectives 5x/0.14NA 10x/0.28NA
Sheet Thickness 2.4 – 21µm FWHM 1.7 – 16µm FWHM
Sheet Length 0.5 – 4mm 0.5 – 2mm
Illumination Direction Bi-Directional
Sheet Formation Galvo mirror sweep

 
Coronal section of Thy1-GFP mouse brain cleared with the PEGASOS method. Sample courtesy of Dr. H. Zhao, Beijing Institute for Brain Research

Shadow Reduction

Infinity Beam is created by a special SLM pattern that converts the Gaussian beam into a beam multiplex. Similar to a flat fan
nozzle, with Infinity Beam the photons approach the specimen from a continuous distribution of angles in the plane of the sheet and are flattened axially in the plane of the objective’s focus. Opaque objects in the specimen that would otherwise cause a shadow artifact instead will have their shadow reduced by the overlap of the continuous angles after the artifact.

Conventional Gaussian Beam

Infinity Gaussian Beam

Galvo Swept Lightsheet

CTLS creates a dynamic lightsheet by sweeping the Gaussian laser beam with a high-speed galvo-controlled mirror. This method allows for a tightly focused uniform lightsheet whose width can be seamlessly expanded or contracted to manage photon budget.

Axial Chromatic Aberration Correction

SlideBook creates an SLM pattern specific to each laser to correct for chromatic shift due to the refractive index of various clearing media. This figure shows epithelial cells lining the vasculature of a mouse brain imaged by 488nm, 561nm & 640nm lasers. Data are viewed in SlideBook’s three-view tool showing XZ, YZ and XY perspectives of the gland before and after axial chromatic correction.

Uncorrected

Corrected

Stages, Chambers and Specimen Holders

High-precision XYZ stages with sub-micron resolution allow for accurate positioning of the specimen. A variety of sample chambers are designed to accommodate specimen size and index matching media. PTFE sample holders are optimized for cleared tissue and are non-reactive with both aqueous and organic clearing solutions.

Small Chamber

52mm(W) 64mm(L) 27mm(H)

Medium Chamber

52mm(W) 64mm(L) 52mm(H)

XL Chamber

88mm(W) 194mm(L) 119mm(H)

Up to 7 different samples, or a whole cleared animal, can be imaged with the multi-specimen holder. Samples are attached to magnetic pedestals that can be stored in index-matching media until they are ready to be imaged.

Temperature Controlled Media

Maintaining constant temperature over time is extremely important to data integrity in multi-hour acquisitions. Small changes in temperature will alter the refractive index of the immersion media, resulting in a shift in the beam above or below the plane of focus. The CTLS specimen chambers are heated and thermally stable to +/- 1ºC.

 

Rendering and Analysis

SlideBook offers a solid set of tools for 3D rendering and analysis. For advanced rendering and analysis 3i offers Aivia. Aivia is an innovative and complete 2-to-5D image visualization, analysis and interpretation platform. Using state-of-the-art algorithm and software architecture, Aivia delivers top performance on critical tasks such as display of large images and analysis of complex biological phenomena. Aivia is powered by a range of machine learning technology for both image segmentation, object classification and novelty detection.

 
A section of the brain – cleared with PEGASOS – is captured with 3i’s Cleared Tissue LightSheet microscope powered by SlideBook software. The data is seamlessly visualized in Aivia and analyzed further with Aivia’s 3D Neuron Analysis recipe. With just a few parameters, the recipe automatically traces all neurons in the volume. The combination of PEGASOS tissue clearing, 3i Cleared Tissue LightSheet, SlideBook software, and powerful Aivia visualization and analysis allows researchers to discover new insights into how the brain functions. Sample courtesy of Dr. Hu Zhao (Texas A&M University) and visualization/analysis courtesy of Aivia (DRVision Technologies).

Cleared Tissue Data Sets

Soft Tissue

 
Kidney
Cdh5-Ai14 TdTomato mouse kidney cleared with PEGASOS. Aivia 3D pixel classifier was used to segment the nephrons. Sample courtesy of Dr. Bo Shen, University Texas Southwestern.
 
Brain
Smooth muscle cells in the arteries, veins, and capillaries of mouse brain cleared with PEGASOS labeled by NG2BacDsRed. Sample courtesy of Dr. Woo-Ping Ge, University of Texas Southwestern Medical Center.
 
Brain
Thy1-GFP mouse brain cleared using the PEGASOS technique. The entire brain was captured at 1µm x 1µm x 4µm resolution using the Hamamatsu Fusion BT sCMOS camera in 1.6 hours and rendered in Aivia.
 
Lung
Mouse lung cleared using CUBIC-R. Aivia 3D segmentation tools were used to identify the morphology of the bronchi and bronchioles (yellow). Sample courtesy of Dr. Luisa Arispe, Northwestern University.

Hard Tissue

 
Femur
Mouse femur cleared with PEGASOS and labeled with Thy1-YFP. Sample courtesy of Dr. Weijing Luo, Texas A&M Health Science Center.
 
Mandible
Mouse mandible cleared with PEGASOS showing vasculature and dentin in the molar teeth reported by endogenous fluorescence cdh5cre. Sample courtesy of Dr. Dian Jing and Dr. Yating Li, Sichuan University, Department of Orthodontics.

Mixed Tissue

 
Hind Paw
Mouse hind paw labeled with Thy-1 YFP and cleared with PEGASOS. Specimen courtesy of Dr. Wenjing Luo, Texas A&M Health Sciences Center.
 
Forelimb
Mouse forelimb cleared with PEGASOS shows cleared carpal, metacarpal, phalanges, and motor neuron fibers expressing tdTomato innervating forelimb muscles.
Spinal Cord
3 different epithelial markers were used to interrogate the vascular morphology of the mouse spinal cord. This specimen is 1.8cm long and cleared with iDISCO. Sample courtesy of Dr. P. Tsoulfas, University of Miami, Miami Project to Cure Paralysis.
 
 
Limb
Axolotl limb from the transgenic line CNP-EGFP, which labels the nervous system. Stained for GFP with an Alexa-Fluor 594 secondary antibody, and nuclear staining with Sytox Green. Image courtesy of Maximina Yun Lab, Center for Regenerative Therapies TU Dresden (CRTD). Clearing protocol: Subiran Adrados C, Yu Q, Bolaños Castro LA, Rodríguez Cabrera LA, Yun MH. (2020) Salamander-Eci: An optical clearing protocol for the three-dimensional exploration of regeneration. Developmental Dynamics. https://doi.org/10.1002/dvdy.264

Specifications

Detection Objectives 0.5x 1.0x 1.5x 2.3x
Correction Plan Apochromat Plan Fluar Apochromat Plan Fluar
Numerical Aperture 0.125 0.25 0.37 0.57
Magnification Range1 0.35x – 5.6x 0.7x – 11.2x 1.1x – 16.8x 1.6x – 25.8x
Lateral Resolution2 2.4µm 1.2µm 0.8µm 0.5µm
Working Distance 114mm 56mm 30mm 11mm
1Zoom optics 0.7x to 11.2x
2Rayleigh criterion
Illumination Objectives 5x/0.14NA 10x/0.28NA
Sheet Thickness 2.4 – 21µm FWHM 1.7 – 16µm FWHM
Sheet Length 0.5 – 4mm 0.5 – 2mm
Axial Sheet Scanning SLM-based with 1 to more than 20 sheets per field of view
Illumination Direction Bi-Directional
Sheet Formation Galvo mirror sweep
Specimen Chambers Small Medium Large (CTLS-XL)
Chamber Size (W,L,H) 52 x 64 x 27mm 52 x 64 x 52mm 88 x 163 x 119mm
Practical Volume 70mL 130mL 1L
Sample Size (X,Y,Z) 12 x 20 x 10mm 12 x 20 x 18mm 20 x 50 x 18mm
Stage Travel (X,Y,Z) 22 x 22 x 22mm 22 x 22 x 22mm 50 x 100 x 50mm
Stage Speed 6mm/s (X,Y,Z) 6mm/s (X,Y,Z) 7mm/s (X,Y) 2mm/s (Z)
Stage Encoder Resolution 8nm 8nm 22nm (X,Y) 6nm (Z)
Refractive Index 1.33 – 1.57
Imaging Speed
Backthinned sCMOS Camera Frame Rate Up to 30fps
6 x 6 x 3mm x 3 wavelengths x 3000 Z planes at 1µm steps, 52ms exposure times – CTLS 80 minutes
6 x 6 x 3mm x 3 wavelengths x 3000 Z planes at 1µm steps, 52ms exposure times – CTLS-XL 120 minutes
Computers Acquisition Analysis
Processor Dual Intel® Xeon® Silver Dual Intel® Xeon® Gold
Processor Cores Dual 10-Core Dual 26-Core
RAM 256GB RAM 256GB RAM
GPU 16GB NVIDIA Quadro RTX5000 24GB NVIDIA Quadro RTX6000
Fast SSD Storage 48TB Fast Acquisition Drive 48TB Fast Acquisition Drive
Software
Acquisition SlideBook with CTLS acquisition console and large data open file format
Analysis SlideBook analysis console with 3D montaging and volume rendering
Advanced Volume Rendering Aivia 3D analysis, segmentation and classification
Lasers Powers up to
405nm 365mW
445nm 365mW
488nm 200mW
514nm 150mW
561nm 150mW
594nm 100mW
640nm 200mW
785nm 300mW

Additional Resources

Brochures

3i Cleared Tissue LightSheet Brochure

Webinars

CTLS Webinar March 31, 2020 – 10AM PT 11AM MT 1PM ET

CTLS Webinar March 31, 2020 – 1PM BST 2PM CEST

Cleared Tissue LightSheet, PEGASOS, and Aivia – May 18, 10AM PDT | 11AM MDT | 1PM EDT

Cleared Tissue LightSheet, PEGASOS Tissue Clearing, and Aivia Visualization and Image Analysis – May 18, 3PM BST | 4PM CEST

Cleared Tissue LightSheet, PEGASOS Tissue Clearing, and Aivia Visualization and Image Analysis – May 20, 10AM CST | 12PM AEST

Find out more about Cleared Tissue LightSheet







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    Phone: +1 (303)-607-9429 x1

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