Rapid pathology of lumpectomy margins with open-top light-sheet (OTLS) microscopy

Abstract

Open-top light-sheet microscopy is a technique that can potentially enable rapid ex vivo inspection of large tissue surfaces and volumes. Here, we have optimized an open-top light-sheet (OTLS) microscope and image-processing workflow for the comprehensive examination of surgical margin surfaces, and have also developed a novel fluorescent analog of H&E staining that is robust for staining fresh unfixed tissues. Our tissue-staining method can be achieved within 2.5 minutes followed by OTLS microscopy of lumpectomy surfaces at a rate of up to 1.5 cm²/minute. An image atlas is presented to show that OTLS image quality surpasses that of intraoperative frozen sectioning and can approximate that of gold-standard H&E histology of formalin-fixed paraffin-embedded (FFPE) tissues. Qualitative evidence indicates that these intraoperative methods do not interfere with downstream post-operative H&E histology and immunohistochemistry. These results should facilitate the translation of OTLS microscopy for intraoperative guidance of lumpectomy and other surgical oncology procedures.

Fig. 1

Schematic of an open-top light-sheet (OTLS) microscope. The OTLS microscope utilizes a solid immersion lens (SIL) and thin oil film to provide wavefront- and index-matching of the illumination and collection beam paths into tissue at a 45-deg angle of incidence. This unique open-top configuration (inset) is versatile for imaging diverse clinical specimens with minimal constraints on size and geometry. The 0.03 illumination NA provides an extended depth of focus (~400 µm) to accommodate for tissue-surface irregularities, specimen tilt, and tissue debris.

Fig. 2

Study design. Freshly excised human breast tissues were inked and bisected immediately after lumpectomy procedures. The bisected surface from one half of the specimen (control specimen) was processed for routine histology (H&E and IHC). The bisected surface from the other half (experimental specimen) was stained and imaged with OTLS microscopy (< 30 minutes), before being processed for routine histology. OTLS surface images were compared to archival H&E histology. In addition, histology images from the experimental and control specimens were compared to show that our tissue-staining and imaging techniques do not interfere with downstream H&E histology and IHC.

Fig. 3

Image acquisition and processing of OTLS microscopy. (a) The dual-channel (nuclear and cytoplasmic channel) OTLS images occupy a combined height (h) of 128 vertical camera pixels (64 pixels per channel, or ~80 µm in tissues). The image height, h = 128 pixels, was selected to optimize the imaging speed while accommodating for surface irregularities, specimen tilt, and tissue debris. (b) Oblique (45-deg) light-sheet images are captured in succession at a sampling pitch of 1.25 μm along the primary tissue-scanning direction, x. The horizontal dimension of each image strip is w = 1.25 mm. (c) The raw light-sheet images are initially stored in a rectangular data cube. During post-processing, this data cube is sheared by 45 deg in the x-z plane to transform the data cube into a trapezoidal data volume, which accurately represents the geometry of the imaged tissue volume. (d) An extended-depth-of-field (EDF) algorithm [84] is applied to extract the irregular surface of the specimen. The two-channel surface-extracted image is then false-colored to resemble H&E histology using an algorithm modified from a recent publication [86]. (e) After false-color processing, adjacent image strips are registered and stitched using an ImageJ grid-stitching algorithm [87].

Fig. 4

A comparison of the image quality between OTLS microscopy of eosin-stained (a) and ATTO 655 NHS-ester-stained (b) fresh breast tissue surfaces. When staining fresh specimens, eosin is not stably bound within the tissue and leaks out of the tissue during imaging (purple arrow), which generates a high background that deteriorates the image contrast. The image panels in (c) and (d) provide image quality comparisons between frozen-section histology with eosin staining, archival FFPE histology with eosin staining, OTLS microscopy with eosin staining, and OTLS microscopy with ATTO 655 NHS-ester staining. Results show that the ATTO 655 NHS ester provides improved contrast for visualizing tissue structures, such as collagen fibers, in comparison to eosin in fresh unfixed tissues. Adipocytes and strands of fibrous tissue with stromal cells remained intact after OTLS microscopy and slide-based “H&E” histology. However, the same tissue structures are heavily distorted in frozen-section “H&E” histology ((d), yellow arrow).

Fig. 5

A fresh breast specimen (1 cm by 1cm by 0.5 cm, (a)) was first stained with SYBR Gold and ATTO 655 NHS ester followed by surface imaging with an OTLS microscope, (b). After OTLS microscopy, the same piece of tissue was submitted for archival FFPE histology (H&E), (c). Panels (d), (e) and (f) show benign breast lobules (red arrow), a duct (purple arrow) and a blood vessel within the adipose tissue (green arrow) that were identified from the OTLS surface image, respectively. The corresponding gold-standard H&E images displaying the same tissue features demonstrate that OTLS microscopy with the SYBR Gold and ATTO 655 NHS ester tissue-staining method can enable rapid and high-quality pathology (1.5 cm²/minute) of a large surgical specimen surface.

Fig. 6

Various microarchitectural features were identified from the OTLS images, including (a) a benign breast lobule where the inset shows individual acini with identifiable lumens, (b) invasive ductal carcinoma (IDC) with Nottingham grade I and (c) IDC with Nottingham grade II, d) ductal carcinoma in situ with comedonecrosis.

Fig. 7

(a) H&E histology and (b) IHC results (ER, PR, and HER2 expression) from different control specimens (untouched by OTLS microscopy methods) as compared to those from OTLS microscopy-processed counterparts, showing that the OTLS methods do not interfere with downstream post-operative H&E histology and IHC analysis.