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.
Highlights
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.
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
Right Side Illumination
Dual-Side Illumination
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 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.
Uncorrected
Corrected
Gland processed using the AdipoClear method. Specimen courtesy of Dr. J. Monks, ob/Gynn-Basic Repro Science, University of Colorado Anschutz Medical Campus
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.
PlanNeoFluar 1.0x / 0.25NA
Apochromat 1.5x / 0.37NA
PlanNeoFluar 2.3x / 0.57NA
Prescan Roadmap
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
Piezo stages with sub-micron resolution allow precise positioning of the specimen. A variety of sample chambers are sized to fit the biology and imaging parameters. Specially designed PTFE sample holders are optimized for cleared tissue and are non-reactive with both aqueous and organic clearing solutions.
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. https://doi.org/10.1002/dvdy.264
Specifications
Resolution
1µm x 1µm x 3µm with 20-tile pattern
Objectives
PlanNeoFluar 1.0x / 0.25NA
Apochromat 1.5x / 0.37NA
PlanNeoFluar 2.3x / 0.57NA
Chambers
25x25x25mm/70ml
25x25x50mm/130ml
50x50x70mm/350ml
Clearing
compatible with organic and aqueous clearing methods
Cameras
sCMOS 2304×2304 6.5µm pixels, 16-bit, back-thinned, 95% QE
Lasers
up to six lines – 405nm, 445nm, 488nm, 514nm, 561nm, 594nm, 640nm
optional 785nm laser for near IR imaging
Stages
X/Y/Z piezoelectric with 22mm travel range, 6mm/s velocity, 8nm step size
Software
SlideBook™ – software for acquisition and GPU-accelerated analysis
Aivia – 5D image visualization, analysis and interpretation
Additional Resources
Brochures
Webinars