Quantitative phase imaging (QPI) modality has been widely adopted in a variety of applications ranging from identifying photomask defects in lithography to characterizing cell structure and tissue morphology in cancer. Traditional QPI utilizes the electromagnetic phase of transmitted light to measure, with nanometer scale sensitivity, alterations in the optical thickness of a sample of interest. In our work, the QPI paradigm is generalized to study depth-resolved properties of phase objects with slowly varying refractive index without a strong interface by utilizing the Fourier phase associated with Fourier-domain optical coherence tomography (FD-OCT). Specifically, based on computing the Fourier phase of light back-scattered by cell nuclei, we have developed nanoscale nuclear architecture mapping (nanoNAM) method that quantifies, with nanoscale sensitivity, (a) the depth-resolved alterations in mean nuclear optical density, and (b) depth-resolved localized heterogeneity in optical density of the cell nuclei. We have used nanoNAM to detect malignant transformation in colon carcinogenesis, even in tissue that appears histologically normal according to pathologists, thereby showing its potential as a pathology aid in cases where pathology examination remains inconclusive, and for screening patient populations at risk of developing cancer. In this paper, we integrate all aspects of nanoNAM, from principle through instrumentation and analysis, to show that nanoNAM is a promising, low-cost, and label-free method for identifying pathologically indeterminate pre-cancerous and cancerous cells. Importantly, it can seamlessly integrate into the clinical pipeline by utilizing clinically prepared formalin-fixed, paraffin-embedded tissue sections.

Phase of an electromagnetic wave propagating through a sample-of-interest is well understood in the context of quantitative phase imaging in transmission-mode microscopy. In the past decade, Fourier-domain optical coherence tomography has been used to extend quantitative phase imaging to the reflection-mode. Unlike transmission-mode electromagnetic phase, however, the origin and characteristics of reflection-mode Fourier phase are poorly understood, especially in samples with a slowly varying refractive index. In this paper, the general theory of Fourier phase from first principles is presented, and it is shown that Fourier phase is a joint estimate of subresolution offset and mean spatial frequency of the coherence-gated sample refractive index. It is also shown that both spectral-domain phase microscopy and depth-resolved spatial-domain low-coherence quantitative phase microscopy are special cases of this general theory. Analytical expressions are provided for both, and simulations are presented to explain and support the theoretical results. These results are further used to show how Fourier phase allows the estimation of an axial mean spatial frequency profile of the sample, along with depth-resolved characterization of localized optical density change and sample heterogeneity. Finally, a Fourier phase-based explanation of Doppler optical coherence tomography is also provided.

Early cancer detection currently relies on screening the entire at-risk population, as with colonoscopy and mammography. Therefore, frequent, invasive surveillance of patients at risk for developing cancer carries financial, physical, and emotional burdens because clinicians lack tools to accurately predict which patients will actually progress into malignancy. Here, we present a new method to predict cancer progression risk via nanoscale nuclear architecture mapping (nanoNAM) of unstained tissue sections based on the intrinsic density alteration of nuclear structure rather than the amount of stain uptake. We demonstrate that nanoNAM detects a gradual increase in the density alteration of nuclear architecture during malignant transformation in animal models of colon carcinogenesis and in human patients with ulcerative colitis, even in tissue that appears histologically normal according to pathologists. We evaluated the ability of nanoNAM to predict "future" cancer progression in patients with ulcerative colitis who did and did not develop colon cancer up to 13 years after their initial colonoscopy. NanoNAM of the initial biopsies correctly classified 12 of 15 patients who eventually developed colon cancer and 15 of 18 who did not, with an overall accuracy of 85%. Taken together, our findings demonstrate great potential for nanoNAM in predicting cancer progression risk and suggest that further validation in a multicenter study with larger cohorts may eventually advance this method to become a routine clinical test

Background: The cell and tissue structural properties assessed with a conventional bright-field light microscope play a key role in cancer diagnosis, but they sometimes have limited accuracy in detecting early-stage cancers or predicting future risk of cancer progression for individual patients (i.e., prognosis) if no frank cancer is found. The recent development in optical microscopy techniques now permit the nanoscale structural imaging and quantitative structural analysis of tissue and cells, which offers a new opportunity to investigate the structural properties of cell and tissue below 200 – 250 nm as an early sign of carcinogenesis, prior to the presence of microscale morphological abnormalities. Identification of nanoscale structural signatures is significant for earlier and more accurate cancer detection and prognosis. Results: Our group has recently developed two simple spectral-domain optical microscopy techniques for assessing 3D nanoscale structural alterations – spectral-encoding of spatial frequency microscopy and spatial-domain low-coherence quantitative phase microscopy. These two techniques use the scattered light from biological cells and tissue and share a common experimental approach of assessing the Fourier space by various wavelengths to quantify the 3D structural information of the scattering object at the nanoscale sensitivity with a simple reflectance-mode light microscopy setup without the need for high-NA optics. This review paper discusses the physical principles and validation of these two techniques to interrogate nanoscale structural properties, as well as the use of these methods to probe nanoscale nuclear architectural alterations during carcinogenesis in cancer cell lines and well-annotated human tissue during carcinogenesis. Conclusions: The analysis of nanoscale structural characteristics has shown promise in detecting cancer before the microscopically visible changes become evident and proof-of-concept studies have shown its feasibility as an earlier or more sensitive marker for cancer detection or diagnosis. Further biophysical investigation of specific 3D nanoscale structural characteristics in carcinogenesis, especially with well-annotated human cells and tissue, is much needed in cancer research.

Objectives:The accurate diagnosis of malignancy from small bile duct biopsy specimens is often challenging. This proof-of-concept study assessed the feasibility of a novel optical technology, spatial-domain low-coherence quantitative phase microscopy (SL-QPM), that assesses nanoscale structural alterations in epithelial nuclei for improving the diagnosis of malignancy in bile duct biopsy specimens. Methods: The SL-QPM analysis was performed on standard histology specimens of bile duct biopsy specimens from 45 patients. We analyzed normal cells with benign follow-up, histologically normal cells with pancreaticobiliary malignancy, and malignant epithelial cells. Results: The SL-QPM–derived nuclear nanomorphology marker can not only distinguish benign and malignant epithelial cells but can also detect features of malignancy in those cells normal by light microscopy with a discriminatory accuracy of 0.90. When combining pathology with SL-QPM, the sensitivity is improved to 88.5% from 65.4% of conventional pathology, while maintaining 100% specificity. Conclusions: SL-QPM–derived nuclear nanomorphology markers represent a novel approach for detecting malignancy from histologically normal-appearing epithelial cells, with potential as an adjunctive test in patients with negative or inconclusive pathologic diagnosis on bile duct biopsy specimens.