Cleared Tissue LightSheet

Cleared Tissue LightSheet Microscope with Laser Stack
Computer monitor with SlideBook Software

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.



Dynamic Lightsheet via Spatial Light Modulator

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.

A “tiled” Gaussian beam tuned to optimize axial resolution and optical sectioning across a 2mm field of view.

CTLS high-speed capture with a 20μm lightsheet (left) and high-resolution capture with a 3μm lightsheet shifted 20 times to produce an optimally resolved image (right).
Ventral tegmental nuclear (VTN) group of the mouse cleared with PEGASOS (Jing et al. (2018). Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Research.)
Sample courtesy of Dr. Hu Zhao (Texas A&M University).

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.

Recording of a live webinar demonstrating the CTLS control module.

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.

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 change from left side to right and back is managed 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
Dual-side illumination showing improved signal-to-noise and capture quality.

Infinity Beam

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, yet 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


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 alveolar buds from a lactating mouse mammary gland excited 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.



Gland processed using the AdipoClear method. Specimen courtesy of Dr. J. Monks, ob/Gynn-Basic Repro Science, University of Colorado Anschutz Medical Campus

Thy1-GFP mouse brain cleared with PEGASOS. Specimen courtesy of Dr. Hu Zhao, Texas A&M University.

Macro Objectives

CTLS incorporates highly corrected apochromat and semi-apochromat macro objectives with high numerical aperture and long working distance that produce superior image data through cleared organs.
PlanApochromat 0.5x/0.125NA
PlanNeoFluar 1.0x / 0.25NA
Apochromat 1.5x / 0.37NA
PlanNeoFluar 2.3x / 0.57NA

Prescan Roadmap

Screen shot of SlideBook's prescan road map of mouse hippocampus

CTLS includes motorized zoom lenses to automatically zoom out and create a 2D 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.

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.


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.

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 for 1.5x, 2.3x
52mm(W) 64mm(L) 27mm(H)

Medium chamber for 1.0x
52mm(W) 64mm(L) 52mm(H)

XL chamber for 0.5x, 1.0x
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.

LaserStack Laser Combiner

3i’s fiber-coupled LaserStack laser combiner allows the combination of up to six lasers covering the entire visible spectrum from 405nm to 785nm at multiple power levels, with an option for near infrared imaging.

Software for Advanced Cleared Tissue Imaging

Control Module

SlideBook’s CTLS control module manages every step in cleared tissue imaging. An intuitive workflow guides users from the selection of imaging resolution to choosing excitation lasers, setting camera exposure, and data acquisition. SlideBook also features 3D reconstruction, 3D rendering, and figure and movie generation with story-board support.

SlideBook’s CTLS control module has been developed with a rich set of features for lightsheet imaging but designed for ease-of-use. It is intuitive yet powerful with features including preview scanning, ROI selection, automated SLM pattern generation, shadow striping attenuation, and axial chromatic aberration correction. SlideBook .SLD files can be accessed via any application supporting Bio-Formats OME, allowing seamless integration with existing workflows in most 3rd-party applications.

The CTLS control module is easy to use – users can expect to set up an experiment in less than 10 minutes. However, the CTLS module is also feature-rich. For example, the galvanometer’s displacement can be optimized effortlessly to maximize signal to noise. Users can set region-by-region overlap to ensure high fidelity 3D montaging with no seams – and without the need for 3rd party stitching software. An SLM Pattern Generator is provided for the intrepid user who would like to experiment with new patterns or to edit patterns that are designed to neutralize axial chromatic aberration and shadowing artifacts.

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).

Powerful Computer Workstation

3i provides high-power computer workstations to control all microscope hardware necessary for acquisition as well as enable processing, segmentation and volume rendering of terabyte (TB) datasets without additional computer resources.  Solid state drives in RAID configurations provide high-speed storage for capture, while traditional high-capacity hard drives provide longer-term storage of datasets. NVIDIA Quadro GPUs work in parallel with the latest Intel Xeon processors for fast stitching, processing and rendering of captured data.

Petabyte Data Storage

3i offers DDN® unified storage systems to allow direct acquisition and analysis, without time-consuming file transfers, at volumes ranging from 500TB to over 2PB. DDN storage systems are an ideal choice for labs and facilities looking to optimize acquisition workflows and/or incorporate data analysis pipelines.

Cleared Tissue Data Sets

Soft Tissue

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.
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.
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.
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

Mouse femur cleared with PEGASOS and labeled with Thy1-YFP. Sample courtesy of Dr. Weijing Luo, Texas A&M Health Science Center.
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

Mouse hind paw labeled with Thy-1 YFP and cleared with PEGASOS. Specimen courtesy of Dr. Wenjing Luo, Texas A&M Health Sciences Center.
Mouse forelimb cleared with PEGASOS shows cleared carpal, metacarpal, phalanges, and motor neuron fibers expressing tdTomato innervating forelimb muscles.
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.


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
GPU 16GB NVIDIA Quadro RTX5000 24GB NVIDIA Quadro RTX6000
Fast SSD Storage 48TB Fast Acquisition Drive 48TB Fast Acquisition Drive
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

Find out more about Cleared Tissue LightSheet



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



    Phone: +1 (303)-607-9429 x2