(C-99) An axially swept nonorthogonal dual-objective (NODO) open-top light-sheet (OTLS) microscope for 3D pathology

Introduction: Nondestructive 3D pathology is an emerging approach for microscopic examination of clinical and preclinical tissues. Compared to conventional 2D histology, nondestructive 3D pathology of optically cleared specimens enables analysis of orders-of-magnitude more tissue volume and accurate characterization of tissue architecture in 3D, all without destructive physical sectioning of valuable samples. Open-top light-sheet (OTLS) microscopy is an imaging platform that is well suited for 3D pathology, providing high throughput imaging without many of the physical constraints on sample size and geometry associated with conventional light-sheet microscopes [1,2]. OTLS systems described to date work well in low-numerical-aperture (NA; < 0.5) and very high-NA (> 1.0) regimes but face practical challenges for high-throughput imaging at the intermediate resolution scales (NA ~0.7) that are most critical for anatomic pathology [3]. Here we describe an optimized OTLS system tailored for rapid imaging of densely labeled clinical specimens.

Materials and Methods: In an OTLS microscope, illumination and collection optics are placed beneath a planar sample holder, allowing tissue specimens of arbitrary size and shape to be imaged from below in a manner akin to a flatbed document scanner. Here we use a non-orthogonal dual-objective (NODO) architecture [4], in which a collection objective is oriented perpendicularly to a planar sample holder while an illumination light sheet is delivered by a second non-orthogonal objective. This approach improves performance at moderate NAs (compared to orthogonal dual-objective OTLS systems) by greatly reducing refractive index-matching requirements between the sample holder and immersion media. This approach also simplifies the microscope design and alignment (compared to single-objective OTLS systems) by decoupling the illumination and collection optical paths.

Results, Conclusions, and Discussions: For intermediate-resolution imaging of densely label clinical specimens (e.g. labeled with fluorescent analogs of conventional hematoxylin and eosin [H&E]), high-quality optical sectioning (high axial resolution) is helpful to distinguish tightly packed cells and nuclei in adjacent 2D imaging planes. To improve optical sectioning ability, we use a novel implementation of axially swept light-sheet microscopy [5] leveraging a voice coil actuator to rapidly scan a higher-NA illumination beam across an increased field of view. We have validated our scanning mechanism through ray-tracing simulations (ZEMAX) and have experimentally demonstrated axial beam scanning at up to 50 Hz over a 750-µm scan range. We are currently assembling a complete OTLS system implementing our approach, which we expect to improve overall image quality in clinical specimens while also significantly increasing volumetric imaging rate.

Acknowledgements (Optional): This work was funded in part by the NIH (R01 EB031002 and R01CA268207) and the NSF (GRFP DGE-1762114).

References (Optional): [1] L. A. Barner, et al., "Multi-resolution open-top light-sheet microscopy to enable efficient 3D pathology workflows," Biomedical Optics Express, 11, 6605 (2020).
[2] A. K. Glaser, et al., "Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues," Nature Communications 10, 1–8 (2019).
[3] K. W. Bishop, et al., "Performance tradeoffs for single- and dual-objective open-top light-sheet microscope designs: a simulation-based analysis," Biomedical Optics Express, 11, 4627–4650 (2020).
[4] A. K. Glaser, et al., "A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues," Nature Methods 19, 613–619 (2022).
[5] K. M. Dean, et al., "Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy," Biophysical Journal 108, 2807–2815 (2015).