Recently, there has been a major thrust to understand biological processes at the nanoscale. Optical microscopy has been exceedingly useful in imaging cell microarchitecture. Characterization of cell organization at the nanoscale, however, has been stymied by the lack of practical means of cell analysis at these small scales. To address this need, we developed a microscopic spectroscopy technique, single-cell partial-wave spectroscopy (PWS), which provides insights into the statistical properties of the nanoscale architecture of biological cells beyond what conventional microscopy reveals. Coupled with the mesoscopic light transport theory, PWS quantifies the disorder strength of intracellular architecture. As an illustration of the potential of the technique, in the experiments with cell lines and an animal model of colon carcinogenesis we show that increase in the degree of disorder in cell nanoarchitecture parallels genetic events in the early stages of carcinogenesis in otherwise microscopically/histologically normal-appearing cells. These data indicate that this advance in single-cell optics represented by PWS may have significant biomedical applications.light-scattering spectroscopy ͉ nanoarchitecture ͉ subdiffusion E xisting knowledge of changes in cell architecture in disease processes is based to a large degree on the histological examination of cells and tissue. On the other hand, it is well accepted that histological and, thus, microarchitectural, aberrations are preceded by molecular, genetic, or epigenetic changes. One may pose a question whether these events are still accompanied by alterations in cell architecture that are histologically undetectable. Indeed, the diffraction limit restricts the resolution of conventional light microscopy to, at best, 200 nm. This is larger than the sizes of the fundamental building blocks of the cell, such as membranes, cytoskeleton, ribosomes, and nucleosomes. Thus, conventional light microscopy is insensitive to changes in nanoarchitecture, which is the fundamental basis of cell organization. It is clear that the fact that a cell is histologically normal may not necessarily be equated with the cell not having nanoscale structural alterations. Cellular alterations in carcinogenesis provide an illustrative and practically important example. The process of carcinoma formation involves stepwise accumulation of genetic and epigenetic alterations in epithelial cells over a time period of many years. Dysplasia, or structural alterations detectable by microscopy, is a relatively late event in this process. From a cancerresearch perspective, it is important to recognize the earlier stages of carcinogenesis that precede histological changes. One can hypothesize that although these genetic/epigenetic aberrations have not yet resulted in histologically apparent changes, they may still be accompanied by architectural consequences that occur at the nanoscale.Therefore, it is of major importance to design optical techniques for inspecting cell nanoarchitecture. One approach to...

A three parameter model based on the Whittle-Matérn correlation family is used to describe continuous random refractive index fluctuations. The differential scattering cross section is derived from the index correlation function using nonscalar scattering formulas within the Born approximation. Parameters such as scattering coefficient, anisotropy factor, and spectral dependence are derived from the differential scattering cross section for this general class of functions.

Existing optical imaging techniques offer us powerful tools to directly visualize the cellular structure at the microscale; however, their capability of nanoscale sensitivity is restricted by the diffractionlimited resolution. We show that mesoscopic light transport theory analysis of the spectra of partial waves propagating within a weakly disordered medium such as biological cells (i.e. partialwave spectroscopy, PWS) quantifies refractive index fluctuations at subdiffractional length scales. We validate this nanoscale sensitivity of PWS using rigorous Finitedifference time-domain (FDTD) simulations and experiments with nanostructured models. We also demonstrate the potential of this technique to detect nanoscale alterations in cells from patients with pancreatic cancer who are otherwise classified as normal by conventional microscopic histopathology.Spectroscopy of elastic scattering is commonly used to probe tissue morphology [1]. However, the sensitivity of light scattering signal to refractive index fluctuations is significantly reduced when the size of the scattering structures falls below the wavelength (~500 nm). Recently, there has been significant interest in understanding biological systems at the nanoscale, which requires measurement of sub-wavelength refractive index fluctuations. According to mesoscopic light transport theory [2][3][4], for an object that is weakly disordered and weakly scattering, it is indeed possible to probe refractive index fluctuations of any length scale including those well below the wavelength [3,4] if one analyzes a signal generated by the multiple interference of 1D-propagating waves reflected from the refractive index fluctuations within the object. The enhanced sensitivity of 1D-propagating waves to sub-wavelength correlation lengths of refractive index fluctuations l c (i.e., l c < wavelength λ) can be understood from the following consideration: while in 3D the scattering coefficient ~(l c / λ) 3 , and, thus, contribution from small length scales is weighted down as , for 1D waves the scattering coefficient is ~ (l c / λ) [5]. The 1D-propagating waves are one of many subset of waves (herein called 1D-partial waves) propagating within a scattering particle. Recently, we reported an optical system [6] capable of isolating 1D-partial waves from different parts of a homogeneous scattering particle. Here we show that the backscattering signal formed by these 1D-partial waves can be detected for heterogeneous objects, such as biological cells, and be used to probe the sub-wavelength refractive index fluctuations.A detailed description of the PWS instrument is given elsewhere [6]. In brief, a broadband light with spatial coherence length <1 μm is focused onto the sample by a low-numerical aperture (NA) objective (Edmund Optics, NA of objective = 0.4, NA of illumination = 0.2, NA of collection = 0.4). The illumination beam diameter (~120 μm) is much larger than biological cells (~8 μm) and is well collimated within a cell located in the waist of the beam. The resulti...

Despite major importance in physics, biology, and other sciences, optical sensing of nanoscale structures in the far-zone remains an open problem due to the fundamental diffraction limit of resolution. We establish that the expected value of spectral variance (Σ̃2) of a far-field, diffraction-limited microscope image can quantify the refractive-index fluctuations of a label-free, weakly scattering sample at subdiffraction length scales. We report the general expression of Σ̃ for an arbitrary refractive-index distribution. For an exponential refractive-index spatial correlation, we obtain a closed-form solution of Σ̃ which is in excellent agreement with three-dimensional finite-difference time-domain solutions of Maxwell's equations. Sensing complex inhomogeneous media at the nanoscale can benefit fields from material science to medical diagnostics.

Exploration of nanoscale tissue structures is crucial in understanding biological processes. Although novel optical microscopy methods have been developed to probe cellular features beyond the diffraction limit, nanometer-scale quantification remains still inaccessible for in situ tissue. Here we demonstrate that, without actually resolving specific geometrical feature, OCT can be sensitive to tissue structural properties at the nanometer length scale. The statistical mass-density distribution in tissue is quantified by its autocorrelation function modeled by the Whittle-Mateŕn functional family. By measuring the wavelengthdependent backscattering coefficient μ b (λ) and the scattering coefficient μ s , we introduce a technique called inverse spectroscopic OCT (ISOCT) to quantify the mass-density correlation function. We find that the length scale of sensitivity of ISOCT ranges from ~30 to ~450 nm. Although these subdiffractional length scales are below the spatial resolution of OCT and therefore not resolvable, they are nonetheless detectable. The subdiffractional sensitivity is validated by 1) numerical simulations; 2) tissue phantom studies; and 3) ex vivo colon tissue measurements cross-validated by scanning electron microscopy (SEM). Finally, the 3D imaging capability of ISOCT is demonstrated with ex vivo rat buccal and human colon samples.