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.
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.
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.
CTLS-XL incorporates a large chamber more than twice the size of standard chambers. The XL may be used to image an entire mouse or large organs roughly 20mm in width and 60mm in length. A 0.5x imaging objective with 114mm of working distance allows for exceptionally fast imaging of very large samples. All CTLS excitation and detection objectives and the smaller glass chambers are compatible with the XL microscope. CTLS-XL represents the ultimate toolkit for cleared tissue imaging of whole animals, large organs, or smaller specimens using chambers that require a modest amount of index matching media.
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
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
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.
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
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.
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.
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.
High-precision XYZ stages with sub-micron resolution allow for accurate positioning of the specimen. A variety of sample chambers are designed to accommodate specimen size and index matching media. PTFE sample holders are optimized for cleared tissue and are non-reactive with both aqueous and organic clearing solutions.
Small chamber for 1.5x, 2.3x
52nm(W) 64mm(L) 27mm(H)
Medium chamber for 1.0x
52mm(W) 64mm(L) 52mm(H)
XL chamber for 0.5x, 1.0x
88nm(W) 194mm(L) 119mm(H)
Up to 7 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.
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.
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.
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.
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.
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.
|Correction||Plan Apochromat||Plan Fluar||Apochromat||Plan Fluar|
|Magnification Range1||0.35x – 5.6x||0.7x – 11.2x||1.1x – 16.8x||1.6x – 25.8x|
|Sheet Thickness||2.4 – 21µm FWHM||1.7 – 16µm FWHM|
|Sheet Length||0.5 – 4mm||0.5 – 2mm|
|Axial Sheet Scanning||SLM-based with 1 to more than 20 sheets per field of view|
|Sheet Formation||Galvo mirror sweep|
|Specimen Chambers||Small||Medium||Large (CTLS-XL)|
|Chamber Size (W,L,H)||52 x 64 x 27mm||52 x 64 x 52mm||88 x 163 x 119mm|
|Sample Size (X,Y,Z)||12 x 20 x 10mm||12 x 20 x 18mm||20 x 50 x 18mm|
|Stage Travel (X,Y,Z)||22 x 22 x 22mm||22 x 22 x 22mm||50 x 100 x 50mm|
|Stage Speed||6mm/s (X,Y,Z)||6mm/s (X,Y,Z)||7mm/s (X,Y) 2mm/s (Z)|
|Stage Encoder Resolution||8nm||8nm||22nm (X,Y) 6nm (Z)|
|Refractive Index||1.33 – 1.57|
|Backthinned sCMOS Camera Frame Rate||Up to 30fps|
|6 x 6 x 3mm x 3 wavelengths x 3000 Z planes at 1µm steps, 52ms exposure times – CTLS||80 minutes|
|6 x 6 x 3mm x 3 wavelengths x 3000 Z planes at 1µm steps, 52ms exposure times – CTLS-XL||120 minutes|
|Processor||Dual Intel® Xeon® Silver||Dual Intel® Xeon® Gold|
|Processor Cores||Dual 10-Core||Dual 26-Core|
|RAM||256GB RAM||256GB RAM|
|GPU||16GB NVIDIA Quadro RTX5000||24GB NVIDIA Quadro RTX6000|
|Fast SSD Storage||48TB Fast Acquisition Drive||48TB Fast Acquisition Drive|
|Acquisition||SlideBook with CTLS acquisition console and large data open file format|
|Analysis||SlideBook analysis console with 3D montaging and volume rendering|
|Advanced Volume Rendering||Aivia 3D analysis, segmentation and classification|
|Lasers||Powers up to|