Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins

Abstract

Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.

Keywords: 3-D deconvolution; H&E staining; fluorescence; lumpectomy; microscopy with UV surface excitation; surgical margin assessment.

Fig. 1

Clinical workflow for intraoperative surgical margin assessment by MUSE. During lumpectomy, the freshly resected tissue specimen is immediately stained with a fluorescent analog of H&E staining. The surgical margin surface of the resected specimen is then imaged with MUSE to guide the resection procedure.

Fig. 2

Schematic of intraoperative MUSE system. (a) A fluorescent analog of H&E staining is used to label fresh tissues within $\sim 5\min$. (b) A MUSE system for comprehensive imaging of fresh specimens. Two LEDs (285-nm wavelength) illuminate the specimen surface at an oblique angle from opposite directions to reduce shadowing artifacts. Fluorescence signal (visible wavelength) is collected and imaged by a $10\times$ ($\mathrm{NA}=0.3$) apochromatic objective and tube lens onto a 2-D detector array. A filter wheel is used to image the two-color channels sequentially. Large-area tiled imaging is achieved by scanning the specimen with a motorized XY stage. (c) At each imaged location (lateral image tile), the piezoactuator scans the specimen vertically to obtain a $z$ stack of images, which allows for 3-D deconvolution (to improve resolution and contrast), surface extraction (to mitigate the effects of surface irregularities), and false coloring to mimic the appearance of gold-standard H&E histology.

Fig. 3

Obtaining and validating a PSF for deconvolution. (a) Illustration of two PSF measurement methods, one of which images subresolution fluorescent beads ($d=\sim 0.2\mu \mathrm{m}$, emission peak at 520 nm) in an agarose phantom (standard method) and another of which images the same beads at the surface of human breast tissue (which adds the effects of tissue scattering). (b) Results showing the FWHM of the PSF theoretically calculated from system parameters (${\mathrm{PSF}}_{\text{theoretical}}$), the average PSF experimentally measured with beads in an agarose phantom (${\mathrm{PSF}}_{\text{beads}}$), and the average PSF experimentally measured with beads at the tissue surface (${\mathrm{PSF}}_{\text{beads}+\text{scattering}}$). (c) Three orthogonal cross-sectional views of ${\mathrm{PSF}}_{\text{theoretical}}$, ${\mathrm{PSF}}_{\text{beads}}$, and ${\mathrm{PSF}}_{\text{beads}+\text{scattering}}$. The colors of the dashed lines correspond to the colors of the data points in (b) and (d) for the $x$, $y$, and $z$ directions (red, green, and blue, respectively). Scale bar: $3\mu \mathrm{m}$. (d) PSF validation results showing the average FWHM of $6\text{-}\mu \mathrm{m}$ beads at a tissue surface without deconvolution, and with deconvolution using three different PSFs. The use of ${\mathrm{PSF}}_{\text{beads}}$ for deconvolution yields the best results regarding resolution and variance. While ${\mathrm{PSF}}_{\text{beads}+\text{scattering}}$ is more accurate, it does not provide improved deconvolution results (as shown by the $p$-value). (b) and (d) Left vertical axis: FWHM in the $x$ and $y$ directions (red and green). Right vertical axis: FWHM in $z$ direction (blue).

Fig. 4

A comparison of image quality for MUSE of breast specimens. Results are shown (a) with deconvolution and surface extraction, (b) with only surface extraction, and (c) of a single frame within a $z$ stack. The green arrows indicate features that are out of focus in a single frame but are brought into focus by surface extraction. The orange arrow points to a cluster of nuclei that are significantly more resolved after deconvolution. These MUSE images are false-colored to mimic H&E histology, based on (e), (g), and (i) a nuclear channel and (d), (f), and (h) a stromal/cytoplasmic channel. (j)–(m) Two zoomed-in regions from the nuclear channel show that deconvolution enables the resolution, contrast, and CNR to be improved. (n) A line profile from region 1 shows that the FWHM of the nuclei are smaller (more resolved) with deconvolution than without. (o) A line profile from region 2 shows that the overall image contrast is also improved by deconvolution.

Fig. 5

A comparison of MUSE image quality with various stromal/cytoplasmic stains. The images show examples of fresh tissues stained with (a) eosin and (b) ATTO 655 NHS ester. (c) With fresh, hydrated specimens, the leakage of eosin, which is poorly bound, results in a pooling effect around the edge of the specimen, and also reduces (e) and (g) image contrast of microstructures. ATTO 655 NHS ester does not exhibit leaking at the (d) tissue edges and provides improved (f) and (h) microstructural contrast. Scale bar: (a) and (b) 1 mm and (c)–(h) $100\mu \mathrm{m}$.

Fig. 6

Human breast tissue imaged with MUSE in comparison to frozen section histology. Large ducts are shown in (a) MUSE and (d) frozen-section histology images. (d) and (e) The frozen sections exhibit optically clear spaces, as indicated by the green arrow) between collagen fibers, which are freezing artifacts that do not appear in the MUSE images. Lobular units are shown in (b) MUSE and (e) frozen section histology images. The yellow arrow in (e) points to a tissue-fold artifact in the frozen section. Adipose tissue is shown in (c) MUSE and (f) frozen-section histology images. The red arrow in (f) indicates distorted adipose tissue and the blue arrow indicates a tear artifact from sectioning the frozen breast tissue. Scale bar: $100\mu \mathrm{m}$.

Fig. 7

MUSE imaging (H&E analog) of benign human breast tissue. After (a) MUSE imaging, a fresh benign human breast specimen ($9\times 10\times 5\mathrm{mm}$), shown in the photo in (c), is submitted for slide-based FFPE H&E histology (b). Images generated by (d) MUSE and (e) slide-based FFPE H&E histology of benign breast lobules are shown with zoomed-in regions highlighting individual acini. Images generated by (f) MUSE and (g) slide-based FFPE H&E histology of breast tissue containing collagen-rich stroma, adipose, and neurovascular bundles, with a zoomed-in region showing a venule. Scale bar: $100\mu \mathrm{m}$ [(d)–(g), first level zoom-in].

Fig. 8

Breast tissue with human DCIS (green arrows) imaged with (a) MUSE, (b) slide-based FFPE histology, and (c) MUSE of an FFPE block face. A photo of the unstained tissue is as shown in (d). Zoomed-in features imaged with (e), (h), and (k) MUSE, (f), (i), and (l) slide-based FFPE histology, and (g), (j), and (m) MUSE of an FFPE block face. (e)–(g) Adipose tissue, (h)–(j) a benign duct, (k)–(m) DCIS. Scale bar: (a)–(c) 1 mm and (e)–(m) $100\mu \mathrm{m}$.

Fig. 9

Human IDC imaged with MUSE. Images generated by (a) MUSE and (e) slide-based FFPE H&E histology are shown of IDC that has invaded a region of adipose tissue. (b) and (c) Images generated by MUSE and (f) and (g) slide-based FFPE H&E histology of IDC that has invaded a region of fibrous stroma. Images generated by (d) MUSE and (h) slide-based FFPE H&E histology of IDC cells with variable nuclear chromatin structure. Scale bar: (a), (b), (e), and (f) $100\mu \mathrm{m}$ and (c), (d), (g), and (h) $50\mu \mathrm{m}$.