Mesoscale volumetric fluorescence imaging at nanoscale resolution by photochemical sectioning.
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| Title: | Mesoscale volumetric fluorescence imaging at nanoscale resolution by photochemical sectioning. |
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| Authors: | Wang, Wei, Ruan, Xiongtao, Liu, Gaoxiang, Milkie, Daniel E., Li, Wenping, Betzig, Eric, Upadhyayula, Srigokul, Gao, Ruixuan |
| Source: | Science. 10/16/2025, Vol. 390 Issue 6770, p1-11. 11p. |
| Subjects: | Fluorescence microscopy, Photochemistry, Microtomy, Axons, Neurodegeneration |
| Abstract: | Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes are physically limited by objective-to-specimen distance, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed a photochemical strategy for spatially precise sectioning of specimens. By combining serial photochemical sectioning with lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelin sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory bulb–wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach. Editor's summary: High-resolution microscopes have a short working distance, making it difficult to see deep within large biological samples such as an intact brain. Slicing the tissue with a blade can reach deeper, but this often distorts or destroys the fine structures that scientists want to study. By embedding a sample in a light-sensitive hydrogel, Wang et al. demonstrated a gentler approach using a precise ray or sheet of light to dissolve or cut away tissue layer by layer. After each layer is removed, the newly exposed surface is imaged, allowing for a complete, high-resolution, three-dimensional reconstruction without damaging physical contact. —Stella M. Hurtley INTRODUCTION: Imaging biological structures at nanoscale resolution deep into large specimens is constrained by the short working distance of objectives used in high-resolution optical microscopes. Hydrogel-based clearing and expansion, while enhancing optical transparency and effective resolution, further push large biological specimens beyond the objective's reach. Mechanical sectioning can circumvent this limit but often introduces distortion, tearing, and loss that complicate large-scale, high-fidelity reconstructions, particularly in hydrogel-embedded and expanded specimens. These limits preclude continuous nanoscale imaging across an intact, whole-mount specimen such as a mammalian brain. RATIONALE: We sought a contact-free, spatially precise alternative to mechanical sectioning that enables nanoscale imaging of whole-mount specimens beyond the microscope's working distance. For this purpose, we combined a photochemical strategy for sample sectioning and sequential on-block optical imaging in a workflow we call VIPS (volumetric imaging via photochemical sectioning). After each imaging cycle, a confined illumination removes a portion of the light-sensitive, hydrogel-embedded specimen to enable a new round of on-block imaging. Iterating these steps, layer by layer, to the desired depth preserves molecular contrast from the anchored biomolecules and yields contiguous volumetric datasets. RESULTS: To implement VIPS, we developed a superabsorbent hydrogel containing a photocleavable cross-linker responsive to ultraviolet excitation. Biological specimens embedded and expanded in this hydrogel underwent rapid, complete photodegradation under either single- or multiphoton illumination. Alternating cycles of on-block lattice light-sheet microscopy (LLSM) and light-sheet photochemical sectioning allowed mesoscale volumetric imaging at nanoscale resolution across two complete mouse olfactory bulbs, one wild-type and one neurodegenerative. A high-performance, petabyte-scale computation pipeline stitched contiguous volumes; reconstructed myelinated and unmyelinated axons; and quantified axon density, tractography, and myelination profiles throughout the bulbs. Olfactory bulb–wide analysis revealed anatomical heterogeneities undetectable in smaller volumes, including a centripetal degeneration pattern of axons in the neurodegenerative bulb. CONCLUSION: VIPS overcomes working-distance limits by coupling iterative photochemical sectioning with high-resolution optical imaging. This noncontact process is compatible with various hydrogel-based clearing and expansion chemistries and circumvents the distortion, sample loss, and protocol limitations inherent to mechanical sectioning. The method integrates readily into existing microscopes for continuous, automated acquisition, enabling nanoscale fluorescence imaging of whole-mount specimens of virtually unlimited size. Together with LLSM and scalable computation, VIPS effectively removes physical sample size as a practical barrier to super-resolution volumetric imaging, allowing for cellular and subcellular studies of intact specimens at petabyte scale and beyond. Remaining challenges include labeling depth and specificity, data storage and computation, and the need for AI systems to interpret massive datasets. Even so, VIPS advances the field from sparse sampling toward quantitative measurements of biological stereotypy and variability in whole specimens. VIPS overview.: VIPS overcomes working-distance limits by light-driven photochemical specimen removal (top). Iterative on-block imaging (imaged volume, dark orange) interleaved with photochemical sectioning produces contiguous volumes, effectively eliminating physical sample size as a constraint. This enables super-resolution imaging of entire mouse olfactory bulbs (center) and comparative, whole-mount analysis of axons and myelin sheaths in wild-type versus neurodegenerative mice (bottom insets). [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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| Abstract: | Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes are physically limited by objective-to-specimen distance, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed a photochemical strategy for spatially precise sectioning of specimens. By combining serial photochemical sectioning with lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelin sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory bulb–wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach. Editor's summary: High-resolution microscopes have a short working distance, making it difficult to see deep within large biological samples such as an intact brain. Slicing the tissue with a blade can reach deeper, but this often distorts or destroys the fine structures that scientists want to study. By embedding a sample in a light-sensitive hydrogel, Wang et al. demonstrated a gentler approach using a precise ray or sheet of light to dissolve or cut away tissue layer by layer. After each layer is removed, the newly exposed surface is imaged, allowing for a complete, high-resolution, three-dimensional reconstruction without damaging physical contact. —Stella M. Hurtley INTRODUCTION: Imaging biological structures at nanoscale resolution deep into large specimens is constrained by the short working distance of objectives used in high-resolution optical microscopes. Hydrogel-based clearing and expansion, while enhancing optical transparency and effective resolution, further push large biological specimens beyond the objective's reach. Mechanical sectioning can circumvent this limit but often introduces distortion, tearing, and loss that complicate large-scale, high-fidelity reconstructions, particularly in hydrogel-embedded and expanded specimens. These limits preclude continuous nanoscale imaging across an intact, whole-mount specimen such as a mammalian brain. RATIONALE: We sought a contact-free, spatially precise alternative to mechanical sectioning that enables nanoscale imaging of whole-mount specimens beyond the microscope's working distance. For this purpose, we combined a photochemical strategy for sample sectioning and sequential on-block optical imaging in a workflow we call VIPS (volumetric imaging via photochemical sectioning). After each imaging cycle, a confined illumination removes a portion of the light-sensitive, hydrogel-embedded specimen to enable a new round of on-block imaging. Iterating these steps, layer by layer, to the desired depth preserves molecular contrast from the anchored biomolecules and yields contiguous volumetric datasets. RESULTS: To implement VIPS, we developed a superabsorbent hydrogel containing a photocleavable cross-linker responsive to ultraviolet excitation. Biological specimens embedded and expanded in this hydrogel underwent rapid, complete photodegradation under either single- or multiphoton illumination. Alternating cycles of on-block lattice light-sheet microscopy (LLSM) and light-sheet photochemical sectioning allowed mesoscale volumetric imaging at nanoscale resolution across two complete mouse olfactory bulbs, one wild-type and one neurodegenerative. A high-performance, petabyte-scale computation pipeline stitched contiguous volumes; reconstructed myelinated and unmyelinated axons; and quantified axon density, tractography, and myelination profiles throughout the bulbs. Olfactory bulb–wide analysis revealed anatomical heterogeneities undetectable in smaller volumes, including a centripetal degeneration pattern of axons in the neurodegenerative bulb. CONCLUSION: VIPS overcomes working-distance limits by coupling iterative photochemical sectioning with high-resolution optical imaging. This noncontact process is compatible with various hydrogel-based clearing and expansion chemistries and circumvents the distortion, sample loss, and protocol limitations inherent to mechanical sectioning. The method integrates readily into existing microscopes for continuous, automated acquisition, enabling nanoscale fluorescence imaging of whole-mount specimens of virtually unlimited size. Together with LLSM and scalable computation, VIPS effectively removes physical sample size as a practical barrier to super-resolution volumetric imaging, allowing for cellular and subcellular studies of intact specimens at petabyte scale and beyond. Remaining challenges include labeling depth and specificity, data storage and computation, and the need for AI systems to interpret massive datasets. Even so, VIPS advances the field from sparse sampling toward quantitative measurements of biological stereotypy and variability in whole specimens. VIPS overview.: VIPS overcomes working-distance limits by light-driven photochemical specimen removal (top). Iterative on-block imaging (imaged volume, dark orange) interleaved with photochemical sectioning produces contiguous volumes, effectively eliminating physical sample size as a constraint. This enables super-resolution imaging of entire mouse olfactory bulbs (center) and comparative, whole-mount analysis of axons and myelin sheaths in wild-type versus neurodegenerative mice (bottom insets). [ABSTRACT FROM AUTHOR] |
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| ISSN: | 00368075 |
| DOI: | 10.1126/science.adr9109 |
