Optical methodology for detecting histologically unapparent nanoscale consequences of genetic alterations in biological cells

Abstract

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...

Quantification of nanoscale density fluctuations using electron microscopy: Light-localization properties of biological cells

“…The preparation of these cells is described elsewhere. 8 Both HT29 cells and their CSK construct cells underwent a standard sample preparation protocol for TEM imaging, including fixing, staining, embedding, sectioning and, finally, performing TEM imaging, as described earlier for the nanoparticles. 7 Figures 3͑a͒ and 3͑b͒ show representative TEM grayscale images of HT29 cells and CSK constructs.…”

“…In our most recent optical experiments, we show that the degree of nanoscale disorder increases with the degree of carcinogenesis for both control and precancerous cells ͑in cell lines, mouse model, and different organs in human studies, such as pancreas, colon, and lung͒. [8][9][10] These nanoscale changes may result from the rearrangements of DNA, RNA, lipids, or proteins. We want to verify and quantify these nanoscale changes as observed in optical studies by TEM.…”

Spectroscopy of Scattered Light for the Characterization of Micro and Nanoscale Objects in Biology and Medicine

“…The method was used to reliably identify genetic alterations in cancer cell lines. 27,28 Clinical studies demonstrated the sensitivity of PWS in detecting pancreatic cancer, colon cancer, and lung cancer, with a novel approach for cancer screening by detecting the field effect of carcinogenesis. 29 An alternate explanation of the nanoscale sensitivity of this measurement was recently presented by making use of the Born approximation and comparing the results with finite-difference time-domain (FDTD) simulations of the microscope with randomly generated refractive index distributions as the numerical samples.…”

Nanoscale refractive index fluctuations detected via sparse spectral microscopy

“…PWS microscopy achieves sensitivity to nanoscale structures within biological cells by using the spectroscopic content of microscope images, and quantitatively measures nanoarchitectural changes in cells associated with carcinogenesis [6,7]. These intracellular, macromolecular alterations are recognizable as some of the earliest indicators of carcinogenesis and are detectable throughout an affected organ, not just at the tumor site, via a phenomenon known as the field effect of carcinogenesis [8,9].…”