Presentation Information
[4FMBS-09-KL]Photodegradable hydrogels for scalable volumetric fluorescence imaging via photochemical sectioning
○Ruixuan Gao1 (1. University of Illinois Chicago (USA))
Keywords:
Hydrogel,Tissue clearing,Tissue expansion,Volumetric fluorescence imaging,Super-resolution microscopy,Spatial biology,Photochemical sectioning
[Purpose] Recent advancements in hydrogel-based tissue clearing and expansion have revolutionized optical nanoscopy of intact biological specimens, enabling molecular-contrast imaging of cellular and subcellular structures. However, current high-resolution fluorescence microscopes are limited by imaging depth, necessitating physical sectioning for whole-mount specimens, regardless of whether they are cleared, expanded, or in their native state.
[Method] To overcome this limitation, we developed Volumetric Imaging of biological specimens via Photochemical Sectioning (VIPS). VIPS utilizes a spatially precise, light-based sample sectioning method called “photochemical sectioning” to achieve volumetric fluorescence imaging across arbitrary sample dimensions. To implement VIPS, we designed and synthesized a class of photodegradable superabsorbent hydrogels that undergo rapid and complete decimation upon UV illumination.
[Results] By combining two-photon photochemical sectioning with volumetric confocal microscopy (“2P VIPS”), we imaged thick mouse brain tissue beyond the objective’s working distance. Furthermore, by combining light-sheet photochemical sectioning with volumetric lattice light-sheet microscopy (“light-sheet VIPS”), we imaged human hippocampus tissue beyond the microscope’s working distance at an effective voxel size of ~22 x 22 x 59 nm. After stitching the imaged volumes, we successfully traced and reconstructed individual axons of as small as ~150 nm in diameter. Finally, by combining light-sheet VIPS with petabyte-scale computation, we imaged and reconstructed axons and myelination sheaths across entire wildtype and neurodegenerative mouse olfactory bulbs at an effective voxel size of ~56 x 56 x 135 nm. Olfactory-bulb-wide analysis of these datasets revealed distinct spatial patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain.
[Consideration] As a non-contact process, VIPS circumvents sample distortion, loss, and protocol constraints often inherent to other sectioning techniques. VIPS integrates seamlessly into existing microscopes for automated acquisition, enabling volumetric fluorescence imaging of whole-mount specimens of virtually unlimited dimensions. While multiple challenges remain, particularly those related to uniform sample labeling, scalable computational infrastructure, and the need for AI-driven interpretation, VIPS represents a conceptual shift from sparse sampling toward in toto quantification of biological stereotypy and variability across tissues, organs, and organisms.
[Conclusion] VIPS overcomes the limited working distance of microscopes through high-resolution optical imaging and spatially controlled photochemical sectioning of biological specimens embedded within a photodegradable hydrogel. Our initial results highlight the potential of VIPS for petabyte-scale super-resolution mapping and beyond, offering unprecedented scalability for large-volume spatial biology.
[Method] To overcome this limitation, we developed Volumetric Imaging of biological specimens via Photochemical Sectioning (VIPS). VIPS utilizes a spatially precise, light-based sample sectioning method called “photochemical sectioning” to achieve volumetric fluorescence imaging across arbitrary sample dimensions. To implement VIPS, we designed and synthesized a class of photodegradable superabsorbent hydrogels that undergo rapid and complete decimation upon UV illumination.
[Results] By combining two-photon photochemical sectioning with volumetric confocal microscopy (“2P VIPS”), we imaged thick mouse brain tissue beyond the objective’s working distance. Furthermore, by combining light-sheet photochemical sectioning with volumetric lattice light-sheet microscopy (“light-sheet VIPS”), we imaged human hippocampus tissue beyond the microscope’s working distance at an effective voxel size of ~22 x 22 x 59 nm. After stitching the imaged volumes, we successfully traced and reconstructed individual axons of as small as ~150 nm in diameter. Finally, by combining light-sheet VIPS with petabyte-scale computation, we imaged and reconstructed axons and myelination sheaths across entire wildtype and neurodegenerative mouse olfactory bulbs at an effective voxel size of ~56 x 56 x 135 nm. Olfactory-bulb-wide analysis of these datasets revealed distinct spatial patterns of axon degeneration and de-/dysmyelination in the neurodegenerative brain.
[Consideration] As a non-contact process, VIPS circumvents sample distortion, loss, and protocol constraints often inherent to other sectioning techniques. VIPS integrates seamlessly into existing microscopes for automated acquisition, enabling volumetric fluorescence imaging of whole-mount specimens of virtually unlimited dimensions. While multiple challenges remain, particularly those related to uniform sample labeling, scalable computational infrastructure, and the need for AI-driven interpretation, VIPS represents a conceptual shift from sparse sampling toward in toto quantification of biological stereotypy and variability across tissues, organs, and organisms.
[Conclusion] VIPS overcomes the limited working distance of microscopes through high-resolution optical imaging and spatially controlled photochemical sectioning of biological specimens embedded within a photodegradable hydrogel. Our initial results highlight the potential of VIPS for petabyte-scale super-resolution mapping and beyond, offering unprecedented scalability for large-volume spatial biology.
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