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 ∕cm 2. 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.

Rapid and High-resolution Imaging of Lumpectomy Margins Using Deep Ultraviolet Fluorescence Scanning Microscopy

“…In contrast, the Xie system requires 5 minutes for staining, 5 minutes to scan a 1.0 cm 2 margin, and 5 minutes for image processing to create uorescent analog of H&E staining. [44] In addition to providing surgeons high contrast images for visual inspection, N/C from DUV images showed signi cant difference between invasive carcinoma and normal breast tissues and thus it may be used as a complementary biomarker for rapid margin detection during BCS. N/C has not been extensively used as a parameter for intraoperative assessment of breast margins.…”

“…The DUV-FSM system described here, like other MUSE devices, [42][43][44] directly images fresh specimens without the need for complex intraoperative radiography and/or pathology processing. This method is extremely simple, easy to use, relatively low in cost, and does not require radiology, pathology or cytology expertise, thereby making it attractive to community hospitals and surgery centers where most BCS procedures are performed.…”

“…They further demonstrated that MUSE could provide microscopic visualization of breast margin surfaces at speeds relevant for intraoperative use. [44] In this study, we investigated the translational potential of MUSE as an intraoperative tool for margin assessment during BCS. Speci cally, we aimed to determine the features of MUSE images that can be used to distinguish fresh, unprocessed malignant from normal/benign breast tissues; the accuracy of the technology; and the speed a lumpectomy specimen can be surveyed.…”

FalseColor-Python: A rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

“…In order to improve throughput, non-destructiveness, sampling extent, and in some cases, to provide 3D information, several slide-free microscopy techniques have recently been explored for use in clinical settings. For example, techniques such as confocal microscopy [1,2], multiphoton microscopy [3][4][5][6], microscopy with UV surface excitation (MUSE) [7][8][9], and structured illumination microscopy (SIM) [10,11] have been explored as slide-free alternatives to frozen-section histology for rapid interoperative guidance. Additionally, light-sheet microscopy technologies, such as open-top light-sheet (OTLS) microscopy [12][13][14], when used in conjunction with tissue-clearing techniques [15], have been explored for slide-free nondestructive 3D pathology.…”

FalseColor-Python: a rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

“…In order to improve throughput, non-destructiveness, sampling extent, and in some cases, to provide 3D information, several slide-free microscopy techniques have recently been explored for use in clinical settings. For example, techniques such as confocal microscopy [1,2], multiphoton microscopy [3][4][5][6], microscopy with UV surface excitation (MUSE) [7][8][9], and structured illumination microscopy (SIM) [10,11] have been explored as slide-free alternatives to frozen-section histology for rapid interoperative guidance.…”