Hyperspectral dark-field microscopy of human breast lumpectomy samples for tumor margin detection in breast-conserving surgery.

Significance: Hyperspectral dark-field microscopy (HSDFM) and data cube analysis algorithms demonstrate successful detection and classification of various tissue types, including carcinoma regions in human post-lumpectomy breast tissues excised during breast-conserving surgeries.

Aim: We expand the application of HSDFM to the classification of tissue types and tumor subtypes in pre-histopathology human breast lumpectomy samples.

Approach: Breast tissues excised during breast-conserving surgeries were imaged by the HSDFM and analyzed. The performance of the HSDFM is evaluated by comparing the backscattering intensity spectra of polystyrene microbead solutions with the Monte Carlo simulation of the experimental data. For classification algorithms, two analysis approaches, a supervised technique based on the spectral angle mapper (SAM) algorithm and an unsupervised technique based on the $K$-means algorithm are applied to classify various tissue types including carcinoma subtypes. In the supervised technique, the SAM algorithm with manually extracted endmembers guided by H&E annotations is used as reference spectra, allowing for segmentation maps with classified tissue types including carcinoma subtypes.

Results: The manually extracted endmembers of known tissue types and their corresponding threshold spectral correlation angles for classification make a good reference library that validates endmembers computed by the unsupervised $K$-means algorithm. The unsupervised $K$-means algorithm, with no a priori information, produces abundance maps with dominant endmembers of various tissue types, including carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma. The two carcinomas' unique endmembers produced by the two methods agree with each other within $<2\%$ residual error margin.

Conclusions: Our report demonstrates a robust procedure for the validation of an unsupervised algorithm with the essential set of parameters based on the ground truth, histopathological information. We have demonstrated that a trained library of the histopathology-guided endmembers and associated threshold spectral correlation angles computed against well-defined reference data cubes serve such parameters. Two classification algorithms, supervised and unsupervised algorithms, are employed to identify regions with carcinoma subtypes of invasive ductal carcinoma and invasive mucinous carcinoma present in the tissues. The two carcinomas' unique endmembers used by the two methods agree to $<2\%$ residual error margin. This library of high quality and collected under an environment with no ambient background may be instrumental to develop or validate more advanced unsupervised data cube analysis algorithms, such as effective neural networks for efficient subtype classification.