Cleared Tissue LightSheet

High Speed High Resolution Imaging of Cleared Tissue and Whole Organs

Cleared Tissue LightSheet (CTLS) is a large field light-sheet microscope designed to image whole organs at high speed. CTLS creates a focused sheet with a narrow waist for better optical sectioning, then uses a spatial light modulator (SLM) to rapidly shift the waist of the sheet along the axis of propagation. A dual excitation setup allows imaging from the right and left sides of the specimen for optimal light-sheet projection throughout. Piezoelectric stages move the specimen in x, y, and z with sub-micron resolution. The result is clear: a 1 cm3 volume can be imaged at up to 1µm x 1µm x 3µm (XYZ) resolution, and a cleared mouse brain can be imaged in as little as 1.5 hours.

The Difference Is Clear

Light sheet microscopy is a powerful technique for imaging large specimens by taking full advantage of emerging tissue clearing methods. The chemistry behind these techniques has advanced to where we can easily penetrate 1, 5 even 10mm into a specimen with a focused sheet of light. In combination with a macro zoom microscope using high NA large field of view lenses, Cleared Tissue LightSheet can image large field sizes with high resolution in short periods of time.

CTLS acquisition is extremely flexible, from ultrafast capture with a 20μm light sheet (left) to high-resolution capture with a 3μm light-sheet shifted 20 times and the resulting 20 sections of best focus tiled to one best-focus image (right).

The movie below shows the 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).

How Tiling Works

The optical sectioning ability of a light sheet is dependent on the thickness of the waist of the focused beam used to create the sheet. The thickness of the waist is directly proportional to the beam length. A thinner waist is generally required for better optical sectioning, but the thinner the waist the shorter the usable length of the beam. Imaging large cleared specimens with a light sheet requires a beam with a long waist matched to the large field of view. The long waist has a correspondingly high thickness, and the result is often poor optical sectioning.

To dramatically improve on this limitation, CTLS uses a spatial light modulator to create a sharply focused beam with a thin waist much shorter than the detector’s field of view. The beam waist is tiled along the axis of propagation and the camera is synchronized to capture one image per tile. The optimal region of each capture is selected and stitched together forming a continuously optimized image. The resulting data has an excellent axial resolution compared to a non-translated focused beam.

Benefits of CTLS

  • Excellent resolution from the macro objectives
  • Excellent optical sectioning from the thin waist of the tiled light sheet
  • Low photobleaching via light sheet as compared to confocal
  • Ultrafast acquisition from large-format macro objectives, large-format sCMOS camera, and digital focus via SLM eliminating the need to move optical elements
  • Fully automated hardware, image acquisition and data stitching via SlideBook software



Allows for optimal sheet penetration across wide specimens and avoidance of opaque structures that may be present on one side but not the other



Macro lenses at 1x/0.25NA and 1.5x/0.37NA can image through any cleared organ with excellent resolution



Sub-micron resolution stages allow precise positioning of the specimen in sample chambers sized to fit the biology



CTLS includes a motorized optical zoom 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 imaging in 3D.


Fiber-coupled laser combiner allows up to six lasers covering the entire visible spectrum at multiple power levels



SlideBook directs all hardware synchronization and data capture, creating 3D datasets at over 1TB ready for analysis and rendering



Available DDN® unified storage systems allow direct acquisition and analysis without time-consuming file transfers for 200TB to over 1PB

SlideBook allows scientists to focus on investigation rather than instrumentation, controlling every aspect of CTLS from hardware configuration to image acquisition, data reconstruction, image processing and 3D visualization. With over 20 years of active development in collaboration with researchers worldwide, SlideBook is intuitive yet powerful with features from visual imaging ROI selection to automated SLM pattern generation and axial chromatic aberration correction. SlideBook SLD files can be accessed via any application supporting Bio-Formats OME, allowing seamless collaboration in any workflow.

Advanced Features

Axial Chromatic Aberration Correction

Leveraging the SLM, SlideBook creates patterns specific to each laser wavelength to correct for axial chromatic aberration. The figure below shows maximum projections of a coronal section of a mouse brain excited with 488nm, 561nm, and 640nm lasers, separated by channel (from left to right, respectively) and merged (far right), before correction (above) and after correction (below).


Automatic Reduction of Shadowing & Striping Effects

Light sheets can be subject to distortion as they encounter objects that have not been cleared, resulting in shadows or striping in the image plane. SlideBook uses the SLM to create a patterned light sheet that interrogates the specimen from 3 different angles to mitigate (and in some instances completely eliminate) these artifacts. The images below show a coronal section of a mouse brain before (left) and after (right) automatic reduction of shadowing/striping effects.

Automatic Reduction Disabled
Automatic Reduction Enabled


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.

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.

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.

Advanced 3D Analysis with Aivia

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).



1 µm x 1 µm x 3 µm



1x/0.25NA, 1.5x/0.37NA



< 1 min/mm3



25mm x 25mm x 25mm (XYZ)



Multiple glass specimen chambers optimized for 1x/0.25NA and 1.5x/0.37NA objectives, with specimen holder.  Custom-shaped specimen holders available upon request.


Organic and aqueous clearing solutions



2048×2048 16-bit sCMOS



LaserStack compact modular laser launch with 488nm and 561nm lasers standard

Up to 4 additional wavelengths upon request



Piezoelectric with sub-micron resolution in X, Y and Z



SlideBook™ software for acquisition and GPU-accelerated analysis

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Cleared Tissue LightSheet


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