AxL Cleared Tissue LightSheet

Axially Swept Light Sheet Microscopy System for Imaging Cleared Specimens

Highlights

Features

Data Sets

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Axially Swept Light Sheet Microscopy System for Imaging Cleared Specimens

AxL Cleared Tissue LightSheet (AxL CTLS) is a fully automated macro zoom microscope with high NA apochromatic objectives and dual-sided light sheet illumination for imaging whole organs to small animals. Custom-designed excitation objectives and patented axially swept light sheet microscopy (ASLM) produce an exceptionally thin, long and uniform lightsheet for large-scale high-resolution imaging. Using a back-thinned sCMOS camera, AxL CTLS images one cubic centimeter in less than a minute.

 

Slide

AxL CTLS Scanner

Proprietary dual-sided light sheet illumination optics
Compact 54 x 60cm footprint
Seamlessly switch imaging modes without changing optics - Axially swept light sheet mode & Ultrafast 3D prescan mode

Back-thinned sCMOS Camera

Multichannel Z Galvo

Automated Zoom Microscope

2304 x 2304 6.5µm pixels
95% quantum efficiency
Rolling shutter mode

High-speed axial chromatic focus adjustment

Motorized 16:1 zoom for optimal pixel sampling

Electrically Tunable Lens

Macro Fluorescence Objectives

LaserStack Laser Combiner

Axial light sheet sweep length of 10mm for large field of view (FOV)

1.0x/0.25NA, WD 56mm
1.5x/0.37NA, WD 30mm
2.3x/0.57NA, WD 10mm

Modular Laser Combiner
Up to 8 lasers
Up to 300mW

Dynamic Galvo-Scanned Light Sheet

Custom Excitation Objectives

Chambers and Specimen Holders

Thin and uniform sheet across a 10mm FOV
Adjustable FOV for faster imaging of smaller specimens

Spherical aberration correction for high-refractive-index immersion media
Flat excitation profile across 1mm to 10mm sheet width
0.14NA diffraction-limited light sheet

Sample chambers in 4 different sizes
Temperature controlled
Chemically-inert sample holders
Clearing solutions ranging from 1.33-1.56 RI
Multi-specimen holder for up to 10 different samples

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

AxL 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.

syglass

syGlass enables 3D and 4D visualization and analysis of SlideBook data in a virtual reality environment.

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

Axially Swept Lightsheet Microscopy

Axially swept lightsheet microscopy (ASLM) – as published in Dean, Fiolka et al., 2015 – scans the light sheet in its propagation direction using high-speed remote focusing synchronized to the rolling shutter readout of an sCMOS camera at the size of the beam waist. This approach creates an exceptionally thin light sheet across a large field of view resulting in images with improved optical sectioning and signal-to-noise. This scanned sheet features a constant laser intensity across the field of view for an evenly illuminated image. The 0.14NA excitation objective creates a 2µm thin waist for superior axial resolution. AxL CTLS is designed to operate optimally across a range of 1.33 to 1.56 refractive indices ensuring compatibility across all available clearing methods.

Low NA

Typical light sheet illumination across the entire field of view.

High NA

Thin static light sheet showing high resolution in the center of the field of view.

High NA ASLM

AxL CTLS light sheet with high resolution across the field of view.

High Speed High Resolution Imaging

The large field of view of AxL CTLS enables ultrafast imaging of whole organs to small animals. An entire mouse can be prescanned in less than 60 seconds, imaged in 20 minutes and high resolution ASLM scanned in 9 hours revealing neuronal connectivity.

Whole Mouse Scan – 20min

Whole Mouse ASLM Scan – 9hrs

whole mouse
Left: Whole mouse imaged in 20min at 0.7x magnification (9.3×9.3x25um voxel size).
Middle: Whole mouse imaged in 9.0h at 1.5x magnification (head displayed only, 4.3×4.3x5um voxel size).
Right: Imaged a subset of the brain at 6.5x magnification (1x1x1um voxel size). Lasers: blue = 488nm, autofluorescence, yellow =  640nm, neurons (Thy1). Courtesy of Dr. Ali Ertürk, Helmholtz Zentrum München.

Zoom

Features for Advanced Cleared Tissue Imaging

Excitation Objectives

Proprietary AxL CTLS excitation objectives deliver an exceptionally thin lightsheet across a wide 10mm field of view and eliminate spherical aberration at high refractive indices. Coupled with macro zoom imaging objectives, AxL CTLS delivers unparalleled optical sectioning with exceptional imaging quality.

AxL CTLS 0.14NA | RI 1.33-1.56

  • Spherical aberration correction for liquid media
  • Optimized for clearing solution refractive index
  • Diffraction-limited light sheet resolution
  • 10mm field of view
mouse embryo
E13.5 mouse embryo cleared with the 3DISCO solvent-based method. Courtesy of Alain Chédotal, Vision Institute.

Dual-Sided Illumination

A pair of excitation objectives focus a lightsheet in front of the camera from the left and right. Either left or right side is selected for optimal sheet penetration across wide specimens, resulting in even illumination and neutralization of shadows caused by opaque structures.

Left Objective

Right Objective

Combined

Shadow Reduction

Because ASLM uses the full numerical aperture of the custom excitation objectives, AxL CTLS produces a full distribution of illumination angles minimizing shadow artifacts dramatically.

Digitally Scanned Light Sheet

AxL CTLS creates a dynamic lightsheet by scanning a Gaussian laser beam using a high-speed galvo-controlled mirror. This method allows for a tightly focused uniform lightsheet whose width can be seamlessly expanded or contracted to cover various fields of views ranging from 0.5mm to 10mm.

Multichannel Imaging Through Z

AxL CTLS can acquire images of samples maintained in aqueous and solvent-based clearing solutions with refractive indices ranging from 1.33 to 1.56. Changes in refractive index require changes in chromatic correction. AxL CTLS chromatically corrects excitation and detection light during image acquisition across wavelengths. An electrically tunable lens adjusts the light sheet focus and a Z galvo correctly sets the image focal plane on the camera.

Below: Epithelial cells lining the vasculature of a mouse brain imaged by 488nm, 561nm and 640nm lasers. Data are shown 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

AxL CTLS incorporates a range of chambers and specimen holders for cleared tissue imaging of whole animals, large organs and smaller specimens. The largest chamber may be used to image an entire mouse or multiple organs while smaller specimens that require a modest amount of index matching media can be imaged in a small, medium or large chamber. High-precision XYZ stages with sub-micron resolution allow for accurate positioning of the specimen. A variety of sample holders optimized for mounting cleared tissue are non-reactive with both aqueous and solvent-based clearing solutions.

Small Medium Large XL
Maximum Sample Dimensions (LWH) 10 x 10 x 10mm 25 x 25 x 20mm 50 x 25 x 20mm 100 x 30 x 30mm
Imaging Media Volume 125mL 250mL 500mL 1000mL
Typical Specimen Zebrafish, Small Mouse Organs Mouse Brain, Rat Brain Mouse Brain with Spinal Cord, Multiple Samples Whole Adult Mouse, Multiple Samples

Sample Holders

With a range of sizes to choose from, these holders are versatile and adaptable to accommodate various sample types. Sample holders are available in various sizes and types including vise-grip style and magnetic mounts.

Up to 10 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 AxL CTLS specimen chambers are thermally stable to +/- 1ºC.

 

Cleared Tissue Data Sets

Mouse embryo
E12 Mouse Embryo cleared with the BABB solvent-based method. Courtesy of Alex Combes and Julie Moreau, Monash University.
 
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.
 
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.
heart and lung
Heart and lung cleared with the iDISCO solvent-based method. Courtesy of Josh Wythe, University of Virginia.
kidney
E18 mouse kidney cleared with the iDISCO solvent-based method. Courtesy of Lori O’Brien, University of North Carolina, Chapel Hill.
Spleen
Mouse spleen cleared with the EZ View aqueous-based method. Courtesy of Juan Cerda, Baylor College of Medicine.
brain
Thy1-GFP mouse brain cleared with the PEGASOS solvent-based method and imaged with the 1.5x / 0.37NA objective in 90 minutes. SlideBook 3D visualization with lookup tables by depth. Courtesy of Dr. H. Zhao, Beijing Institute for Brain Research.
 
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.
 
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
Cortico-spinal projections showing neurons in the hypothalamus connecting to the spinal cord. Mouse brain cleared with the 3DISCO solvent-based method. Courtesy of Dr. P. Tsoulfas, University of Miami, Miami Project to Cure Paralysis.
brain
Mouse cerebellum cleared with the iDISCO solvent-based method. Courtesy of Jessica Verpeut, Arizona State University.
brain
Mouse cortical pyramidal cells cleared with the iDISCO solvent-based method. Courtesy of Jessica Verpeut, Arizona State University.
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.
mouse embryo
E13.5 mouse embryo cleared with the 3DISCO solvent-based method. Courtesy of Alain Chédotal, Vision Institute.
mouse embryo
E13 mouse embryo cleared with the 3DISCO solvent-based method. Courtesy of Ted Usdin and David Leopold, National Institutes of Health (NIH).
 
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.
 
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.
Hind Limb
Mouse hindlimb with femur and tibia cleared with the EZ View aqueous-based method. Courtesy of Josh Wythe, Gabrielle Largosa and Ted Ahn, University of Virginia.
zebrafish
Whole zebrafish brain cleared with the iDISCO+ solvent-based method. Courtesy of Liz Haynes, The Morgridge Institute for Research.
 
 
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

Additional Resources

Brochures

AxL Cleared Tissue LightSheet

Webinars

 

AxL Cleared Tissue LightSheet – North America: February 28, 2024

Find out more about Cleared Tissue LightSheet









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

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