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