Fourier phase based depth-resolved nanoscale nuclear architecture mapping for cancer detection

Highlights

• FD-OCT based extension of quantitative phase imaging to the reflection mode.

• Fourier phase based modeling of refractive index of weakly scattering objects.

• Optical system for measuring refractive index based nuclear structural properties.

• Application of the method to detect early-stage carcinogenesis.

Abstract

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.

Introduction

The past decade has seen the emergence of quantitative phase imaging (QPI) as a powerful imaging modality [1] with applications in a wide variety of areas ranging from lithography [2] to biology [3]. In the latter case, QPI computes the complex amplitude of light transmitted through weakly scattering biological specimens to obtain their optical thickness. Such measurements have been used to study live cell dynamics [4], cell volume and dry mass [5], and tissue architecture [6], [7] among other things [8]. A distinct advantage of QPI is its ability to measure alterations in optical thickness with nanoscale sensitivity, thereby providing a mechanism to visualize and contrast small structural changes in phase objects.

The underlying physics of QPI states that quantitative phase of QPI results from the coherent accumulation of electromagnetic phase of normally incident light wave as it propagates through the entire sample depth. A different physics emerges, if the focus is shifted from transmitted light to back-scattered light. Since the back-scattered waves are generated from different depths along the axial length of the sample, the notion of a single electromagnetic phase is no longer applicable. In this scenario Fourier-domain optical coherence tomography (FD-OCT) has successfully generalized the QPI paradigm to the reflection mode by computing the phase associated with the Fourier transform of the spectral interference signal that results from interference between the back-scattered waves and reference waves for a broadband light source [9], [10], [11]. The resulting Fourier phase is depth-resolved due to the coherence gate imposed by the light source. When a strong interface of interest is present within the sample, it is capable of measuring sub-resolution shifts in the interface location within the coherence gate with high sensitivity [9]. As a result, it forms the basis for various FD-OCT-derived imaging modalities such as optical coherence phase microscopy [10], [12], [13], doppler-OCT [15], and phase-sensitive OCT [16], [17].

We have recently shown that for weakly scattering samples with slowly varying refractive index and without any strong interfaces, Fourier phase provides access to structural properties of the sample beyond measuring sub-resolution shifts. Specifically, it provides access to the joint-estimate of sub-resolution offset and the depth-resolved mean spatial frequency of the coherence-gated sample refractive index [11]. The former is a generalization of sub-resolution shift in the depth location of a strong interface to the setting where no strong interfaces are present. It measures the offset of the weighted-center of the sample refractive index within the coherence gate – conceptually akin to the center-of-mass of a body – from the depth location where the coherence gate is centered. The mean spatial frequency estimates two depth-resolved structural properties of the sample within the coherence gate: (a) alterations in mean refractive index, and (b) localized heterogeneity of the refractive index.

Based on this generalized understanding of Fourier phase, we have developed the method of nanoscale nuclear architecture mapping (nanoNAM) [18], which computes with nanoscale sensitivity the depth-resolved mean spatial frequency of the refractive index of a cell nuclei. nanoNAM does so through the marginalization of Fourier phase along the sub-resolution offset dimension. As a result, nanoNAM measures (a) depth-resolved alterations in mean nuclear optical density of the cell nucleus, and (b) depth-resolved localized heterogeneity in optical density of the cell nucleus. We use the term optical density to emphasize the internal architecture of the cell nucleus in terms of the optical property of its nuclear refractive index.

It should be noted that a phase object can be structurally described through the spatial distribution of its refractive index. The physics behind this interpretation of the cell nucleus is the basis for the ability of nanoNAM to measure depth-resolved properties of the nuclear refractive index distribution with nanoscale sensitivity [11], [19]. This ability, in turn, is significant in its potential for describing subtle structural changes of cell nuclei during biological processes involved in disease pathology. One important example is the pathogenesis of cancer. It has been well-established that genetic and epigenetic disruptions play an important role in all stages of cancer development including in histologically normal-appearing precursor cells [20], [21] in early stage carcinogenesis. One key consequence of this dysregulation is the change in chromatin structure and spatial organization [22]. These changes eventually manifest as alterations in nuclear architecture of cancer cells, with enlarged nuclei size, irregular shape, and hyperchromasia being the most noticeable and universal features [23], [24]. However, in early stage carcinogenesis, these neoplastic transformations in nuclear architecture are often too subtle to be visible. Current state-of-the-art in cancer diagnosis is based on pathologists visualizing morphological abnormalities through conventional microscopic imaging of hematoxylin and eosin (H&E)-stained tissue sections; it is not sensitive enough to detect the subtle nuclear architectural changes in cells undergoing early-stage neoplastic transformation that appear histologically normal. Conventional QPI modalities, and other more advanced super-resolution imaging modalities have drawbacks that limit their use, especially in routine clinical settings (see Section 2.2.4 for details). As a result, the limitation of conventional pathology leads to indeterminate diagnosis in some cases, missed early-stage cancers in others, and limited ability to assess cancer risk. In such cases nanoNAM provides a unique ability to measure and track structural alterations in the cell nuclei, as the cells undergo malignant transformations during early stage carcinogenesis.

In the next section, we discuss Fourier phase in the context of weakly scattering samples without strong interfaces, and employ a mathematical model to show how nanoNAM method derived from Fourier phase measures nuclear architectural properties of depth-resolved alterations in mean nuclear optical density and localized heterogeneity in nuclear optical density. Simulations are also presented to demonstrate the underlying concepts. In Section 3 we introduce the nanoNAM optical system and extensively discuss its instrumentation and sample preparation protocols. Section 4 details the nanoNAM processing steps that compute the above-mentioned nuclear architectural properties from the spectral interference signal measurements. All practical implementation details are discussed. In Section 5, we give an example of the significance of nanoNAM in assessing colorectal cancer risk in patients with ulcerative colitis (UC), an inflammatory bowel disease associated with chronic colonic inflammation. Concluding remarks discussing current limitations of nanoNAM, and its future direction are presented in Section 6.