The 3Di™ Hybrid Open-Top Light-Sheet Microscope: multi-scale 3D imaging without compromise
If you work with intact tissues and want true spatial context, you usually face a trade-off: whole-sample speed versus sub-cellular detail, flexible mounting versus optical performance.
Alpenglow Biosciences’ 3Di™ Hybrid Open-Top Light-Sheet (HOTLS) microscope is built to eliminate that trade-off, delivering rapid whole-tissue scouting and targeted high-resolution zoom on the same instrument, with an open-top design that simplifies and makes repeatable sample handling.
What “hybrid open-top light-sheet” means, and why it matters
Conventional light-sheet systems often constrain specimen size and mounting because the objectives surround the sample. Open-top systems invert this arrangement, with the optics below the specimen. Alpenglow’s hybrid architecture combines two light-sheet paths on a single platform: an orthogonal open-top path for fast, wide-field scouting and a non-orthogonal dual-objective path for sub-micrometer zoom. This pairing lets you screen large volumes quickly, then re-image selected regions at higher resolution without moving to a different microscope.
The hybrid concept was introduced and validated in Nature Methods in 2022. The paper describes the non-orthogonal dual-objective (NODO) path, remote refocusing to correct a tilted light sheet, and multi-scale workflows, all demonstrated on intact mouse brain and metastatic lesions.
Before that, the team established the open-top foundation with multi-immersion optics for cleared tissues in Nature Communications in 2019, addressing practical mounting, compatibility with many clearing protocols, and high-throughput imaging. These engineering choices are core to today’s product, the 3Di™.
What you can expect from the 3Di™ HOTLS in practice
Two imaging modes on one instrument
Scout (ODO path) for rapid, low-resolution whole-sample surveys and navigation.
Zoom (NODO path) for localized, high-resolution imaging of regions identified during Scout.
On the commercial system, you move between modes in the same session, using a single user interface named LUMI.
“LUMI is the microscope’s GUI for setup, acquisition, and live monitoring. It supports:
- Intuitive scan setup, so operators configure trays, tiles, and channels quickly.
- Simultaneous live previews in Scout and Zoom, which helps position ROIs and verify focus before committing to long scans.
- A clean, easy interface usable by new and advanced users, with a “see it working” preview”
Large imaging volume with open-top convenience
Tiled imaging over up to ~12 × 7.5 × 1 cm volumes, suitable for multi-sample trays, large organs, or arrays of biopsies.
Open-top holders keep cleared specimens stable by gravity and light compression, while separating immersion and clearing solutions for easier handling.
Resolution and optics at a glance
Zoom path pixel sampling on the order of ~170 nm per pixel, with collection NA up to 0.7 for sub-micrometer detail.
Scout path wide field surveying with pixel sampling around ~2 µm per pixel, used to find and queue ROIs for Zoom.
Multi-immersion optics spanning refractive indices ~1.33 to 1.56 to match common clearing media. 3Di™ HOTLS Alpenglow
Throughput and automation
Motorized stage for raster tiling across large trays, including multi-well plates and arrays like “8+ mouse brains” in a single run.
Fully automated multi-sample workflows, with cloud upload, flat-fielding, stitching, registration, and AI-ready outputs. 3Di™ HOTLS Alpenglow
From imaging to answers: 3Di™, 3Dm™, and 3Dai™
Alpenglow positions the microscope within an end-to-end platform:
3Di™ for 3D images acquisition,
3Dm™ for data management and correction,
3Dai™ for quantitative analysis, including counts, shapes, distances to vessels, segmentation, and spatial correlations.
This pipeline is designed for non-destructive 3D pathology, preserving tissue for downstream assays and enabling larger, decision-grade volumetric datasets.
Typical samples and clearing compatibility
The instrument was engineered around cleared tissue imaging across protocols with different refractive indices. The open-top layout accepts whole organs, biopsies, organoids, and multi-well plates, with customizable holders to match geometry and workflow. The multi-immersion objective design and open-top mounting were created specifically to accommodate this variety.
A few example workflows among many applications
Whole-organ survey with targeted ROI quantification
Scout the full organ, mark ROIs, then Zoom to quantify axonal density, vascular branching, or tumor invasion margins in situ, as demonstrated in the 2022 Nature Methods paper on intact brain and brain metastasis models. NatureBiopsy-scale 3D pathology with preserved tissue
Image intact biopsies, run 3Dai™ analyses for counts, shapes, and distances, then keep the specimen for sequencing or IHC. The non-destructive approach and data-processing pipeline are described in the 2021 Perspective. NatureHigh-throughput cleared tissue screens
Load multi-sample trays, run automated tiling and cloud correction, and export stitched volumes for downstream AI. This high-throughput use case builds directly on the multi-immersion open-top design reported in 2019. Nature
Key specifications and capabilities
Imaging volume: up to ~12 × 7.5 × 1 cm tiled volume per run.
Dual paths: Scout for speed, Zoom for sub-micrometer detail.
Pixel sampling: ~2 µm per pixel in Scout, ~170 nm per pixel in Zoom.
Collection NA: up to 0.7 on the Zoom path.
Lasers: standard 405, 488, 561, 638 nm (customizable)
Multi-immersion range: n ≈ 1.33 to 1.56.
End-to-end pipeline: integrated upload, corrections, registration, and quantitative 3D analytics.
For a product overview and screenshots of the LUMI interface, see the HOTLS page: https://www.alpenglowbiosciences.com/3di-hotls-microscope
How it fits into your lab or program
Alpenglow supports different engagement models, from imaging services and pilot projects to placing a full solution on-site with training and support. The FAQ and site overview describe the combined hardware, software, and services approach for research, translational studies, and early clinical investigations.
Where to learn more
Product page: 3Di™ Hybrid Open-Top Light-Sheet Microscope.
Brochure with specs and workflow diagrams, including imaging volume, multi-immersion optics, Scout and Zoom details, and analysis examples. 3Di™ HOTLS Alpenglow
Peer-reviewed papers that underpin the system
Glaser et al., Nature Methods 2022, hybrid HOTLS with NODO and remote refocusing, multi-scale imaging demonstrations. Nature
Glaser et al., Nature Communications 2019, multi-immersion open-top design for cleared tissues and high-throughput workflows. Nature
Liu, Glaser, Madabhushi, Nature Biomedical Engineering 2021, Perspective on non-destructive 3D pathology and its computational stack. Nature
Use-case posts that contrast 3D with 2D: Alpenglow Biosciences
The 3Di™ HOTLS delivers what many groups have wanted from light-sheet imaging, a practical way to combine whole-sample context with sub-cellular detail, while keeping the tissue intact and the workflow scalable. If your questions require complete morphology, true spatial relationships, and quantitative 3D readouts, a hybrid open-top approach is worth serious consideration.
