title: Assessment of breast pathologies using nonlinear microscopy.
abstract: Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.
authors.affiliation: Department of Electrical Engineering and Computer Science and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Yuankai K
authors.initials: YK
authors.lastname: Tao
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: Dejun
authors.initials: D
authors.lastname: Shen
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: Yuri
authors.initials: Y
authors.lastname: Sheikine
authors.affiliation: Department of Electrical Engineering and Computer Science and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Osman O
authors.initials: OO
authors.lastname: Ahsen
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: Helen H
authors.initials: HH
authors.lastname: Wang
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Schmolze
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: Nicole B
authors.initials: NB
authors.lastname: Johnson
authors.affiliation: Imaging Systems, Thorlabs, Inc., Sterling, VA 20166; and.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Brooker
authors.affiliation: Advanced Imaging Group, Thorlabs, Inc., Newton, NJ 07860.
authors.forename: Alex E
authors.initials: AE
authors.lastname: Cable
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
authors.forename: James L
authors.initials: JL
authors.lastname: Connolly
authors.affiliation: Department of Electrical Engineering and Computer Science and Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA 02139; jgfuji@mit.edu.
authors.forename: James G
authors.initials: JG
authors.lastname: Fujimoto
mesh_terms: D001940:Breast / Q000473:pathology*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000473:pathology*; D018270:Carcinoma, Ductal, Breast / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000473:pathology; D005260:Female; D006801:Humans; D008853:Microscopy / Q000379:methods*; D009361:Neoplasm Invasiveness; D017711:Nonlinear Dynamics; D012680:Sensitivity and Specificity
keywords: breast cancer; imaging; nonlinear microscopy; pathology