Light-Sheet Imaging of Cleared Samples

From mapping neuronal networks across entire mouse brains to visualizing developing nerves in intact embryos, imaging of cleared samples enables researchers to study complex biological systems at unprecedented scale and resolution. The examples below highlight how researchers can study cells, tissues, and biological networks within their full anatomical context across large specimen volumes.

For a deeper look at these and other use-cases, you can see our most popular resources related to light-sheet imaging of cleared samples here.

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In this example, a mouse brain cleared with the Universal Tissue Clearing Kit and labeled for tyrosine hydroxylase is shown in four views of the same sample at progressively higher magnification and higher resolution.

Presenting the dataset across multiple scales allows researchers to examine the overall organization of the sample while also exploring finer details within specific regions of interest.

The Universal Tissue Clearing Kit, Bruker's light-sheet microscopes, and the Acquifer HIVE centralized data storage and computing solution are complementary technologies that support sample preparation, imaging, and data processing within a unified workflow. Together, these technologies support the generation and management of high-quality 3D datasets while helping researchers align each stage of the cleared tissue imaging process.

Figure 1: Mouse brain cleared with the Universal Tissue Clearing Kit, IF labeling of tyrosine hydroxylase. Imaged with the Bruker Luxendo LCS SPIM.

In this example, a cleared mouse brain labeled for tyrosine hydroxylase is shown at multiple scales, including a maximum intensity projection of the image stack, a corresponding single optical slice, and a magnified view of a selected region of interest.

Together, these visualizations illustrate how cleared tissue imaging can support the exploration of large three-dimensional datasets while maintaining access to individual image planes and higher-magnification views. This multi-scale approach allows features of interest to be viewed within the context of the broader sample while also highlighting localized details.

Figure 2: Mouse brain cleared with the Universal Tissue Clearing Kit, IF labeling of tyrosine hydroxylase. Shown as a maximum intensity projection (MIP) of the image stack (top), a single optical slice at the same view and magnification (middle), and a magnified view of a selected region of interest (bottom). Imaged with the Bruker Luxendo MuVi SPIM, 20X CS objective.

In this example, microglia within a cleared mouse brain are visualized using a 20× tiled acquisition on the Bruker Luxendo MuVi SPIM. Combining tissue clearing with high-resolution light-sheet imaging enables researchers to capture large three-dimensional datasets while maintaining detailed visualization of cellular features throughout the sample.

Figure 3: Mouse brain cleared with the Universal Tissue Clearing Kit. Imaged with the Bruker Luxendo MuVi SPIM, 20X tiled acquisition, Nikon 20X 1.0NA objective.

In this example, pyramidal neurons are imaged throughout a whole cleared mouse brain, allowing these cells to be visualized within the global context of the entire organ. By combining tissue clearing with light-sheet fluorescence microscopy, researchers can generate high-resolution datasets from large specimens that exceed the microscope's field of view through tiling and stitching.

This approach supports the study of neural architecture within the context of the entire brain, providing insights that are difficult to obtain using conventional section-based imaging.

Figure 4a: Overview of an entire mouse brain (anterior-posterior axis horizontally, dorsal-ventral axis vertically). Scalebar: 1000 μm.

Figure 4b: The achieved isotropic resolution of pyramidal neurons. Scalebar: 50 μm.

Courtesy of: Dan Zhang, Ph.D., Core Facility of Center of Biomedical Analysis at Tsinghua University, Beijing, China.

In this example, GFP-labeled neurons within a CUBIC-cleared brain are visualized, allowing individual neuronal structures, including axons and dendrites, to be imaged and traced within the surrounding tissue. The image is displayed as a maximum-intensity projection with color coding based on depth, with warmer colors representing structures nearer the top of the image and cooler colors representing structures deeper within the sample.

This visualization highlights how three-dimensional spatial information can be preserved and interpreted within a single projected image.

Figure 5: Neuronal network (stained with GFP) of a cleared mouse brain. Color-coded depth representation: maximal depth displayed 1.23 mm. Imaged with the MuVi SPIM.

Courtesy of: Montserrat Coll Lladó, European Molecular Biology Laboratory (EMBL), Barcelona, Spain

In this example, developing nerves are visualized throughout an intact mouse embryo, providing a three-dimensional view of nervous system formation across the entire organism. By combining whole-embryo clearing with fluorescent labeling, researchers can visualize neural structures in their native anatomical context rather than relying on individual tissue sections. Preserving this spatial context provides valuable insights into nervous system formation and connectivity during embryonic development.

Viewing these processes within the intact embryo helps reveal relationships and patterns that may not be apparent when tissues are studied in isolation.

Figure 6: Developing nerves in a whole mouse embryo. Tiled image (3 x 4) acquisition. Scalebars: 1 mm. Imaged with the LCS SPIM.

Courtesy of: James Muller, MSKCC, New York, USA.

In this example, the intricate branching patterns of mouse lungs and blood vessels are visualized throughout an intact organ, revealing their three-dimensional organization. By combining tissue clearing with light-sheet imaging, researchers can examine tissues and organs in their entirety and gain in-depth insights into their geometric patterning.

Viewing these structures within the context of the entire organ provides a more complete understanding of how tissues and vascular networks are organized.

Figure 7: Light-sheet images of a lung taken with the LCS SPIM.

Courtesy of: Ayelen Melina, Santamans Recchini, and Gaudalupe Sabio Buzo, Stress kinases in Diabetes, Cancer and Cardiovascular Disease Laboratory and Unit of Microscopy and Dynamic Imaging, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid.

In this example, developing nerves and differentiating muscles are visualized throughout a cleared mouse head, revealing their organization within a large, intact specimen. By combining fluorescent labeling with tiled light-sheet imaging, researchers can capture high-resolution datasets across fields of view that extend beyond the size of a single image acquisition. This enables large anatomical structures to be imaged in their entirety while preserving fine structural detail.

Examining these tissues within an intact three-dimensional volume helps researchers interpret complex anatomical relationships that can be difficult to reconstruct from individual tissue sections.

Figure 8: Cleared mouse head, labeled with anti-tuj1 (green) to mark developing nerves and with anti-desmin (red) to mark differentiating muscles. Tiled image (6 × 5). Imaged with the MuVi SPIM.

Courtesy of: Glenda Comai, Institut Pasteur, Paris, France

In this example, a cleared mouse lymph node is reconstructed in three dimensions using Bruker's cleared sample imaging workflow. By combining tissue clearing with light-sheet fluorescence microscopy, researchers can image intact biological specimens and generate detailed volumetric datasets while preserving critical biological context. The resulting reconstruction demonstrates how large tissue volumes can be visualized in their entirety and explored within a three-dimensional framework.

Preserving the complete tissue architecture enables researchers to examine spatial relationships throughout the entire lymph node, supporting studies of lymphoid organization, vascular architecture, and tissue remodeling in health and disease.

Figure 9a: Cleared mouse lymph node. High endothelial venules (642 nm, red) and autofluorescence (488 nm, green) to visualize surrounding tissue. Imaged with the MuVi SPIM.

Figure 9b: BABB-cleared mouse lymph node. High endothelial venules (red) and autofluorescence (green) to visualize surrounding tissue.

Courtesy of: Jens Stein, University of Bern, Bern, Switzerland

Based on cleared sample imaging workflows and needs, we recommend that interested users start by exploring the systems highlighted below.