Cleared Tissue LightSheet

High Speed High Resolution Imaging of Cleared Tissue and Whole Organs

Cleared Tissue LightSheet (CTLS) is a large field 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 lightsheet 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.

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 and 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).

How it works

Lightsheet 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.

The optical sectioning ability of a light sheet is dependent on the waist of the focused beam, and the waist of the beam is inversely proportional to its length. Imaging large, cleared specimens typically requires long beams with a thick waist producing poor optical sectioning. To dramatically improve optical sectioning, CTLS uses an SLM programmed by SlideBook software to create short beams with narrow waists that are rapidly translated across the field of view. The resulting data has an excellent axial resolution compared to a non-translated focused beam.

Benefits of CTLS

  • excellent x/y resolution from the macro objectives
  • excellent axial resolution from the thin waist of the light sheet
  • low photobleaching due to the use of lightsheet vs. confocal
  • fast acquisition due to large format macro objectives and a large format sCMOS camera

Key Features

DUAL LEFT AND RIGHT ILLUMINATION

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

 

HIGH NA / LARGE FIELD OF VIEW / LONG WORKING DISTANCE OBJECTIVES

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

 

PIEZO STAGES DRIVING MULTIPLE SAMPLE CHAMBERS

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

LASERSTACK LASER COMBINER

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

 

GPU-OPTIMIZED SLIDEBOOK SOFTWARE

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

 

LARGE DATA SOLUTIONS

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

Mouse forelimb shows cleared carpal, metacarpal and phalanges and motor neuron fibers expressing tdTomato innervating forelimb muscles. Cleared with PEGASOS, imaged on Cleared Tissue LightSheet (CTLS), and rendered with Aivia.
Whole brain of thy1-GFP adult mouse cleared with PEGASOS, imaged on Cleared Tissue LightSheet (CTLS), and rendered with Aivia.

Specifications

RESOLUTION

1 µm x 1 µm x 3 µm

 

OBJECTIVES

1x/0.25NA, 1.5x/0.37NA

 

IMAGING SPEED

< 1 min/mm3

 

SAMPLE TRAVEL RANGE

25mm x 25mm x 25mm (XYZ)

 

SPECIMEN CHAMBERS

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.

COMPATIBLE CLEARING METHODS

Organic or aqueous clearing solutions

 

CAMERA

2048×2048 16-bit sCMOS

 

LASER LINES

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

Up to 4 additional wavelengths upon request

 

XYZ TRANSLATION STAGE

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

 

SOFTWARE

SlideBook™ software for acquisition and GPU-accelerated analysis

Find out more about
Cleared Tissue LightSheet






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