Jeremy D. Rogers

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

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.

Toward the development of miniaturized imaging systems for detection of pre-cancer

In this paper, we describe the progress toward the development of miniaturized imaging systems with applications in medical imaging, and specifically, detection of pre-cancer. The focus of the article is a miniature, optical-sectioning, fluorescence microscope. The miniature microscope is constructed from lithographically printed optics and assembled using a bulk micro-machined silicon microoptical table. Optical elements have been printed in a negative tone hybrid glass to a maximum depth of 59 /spl mu/m and an rms surface roughness between 10-45 nm, fulfilling the requirements of the miniature microscope. Test optical elements have been assembled using silicon-spring equipped mounting slots. The design of silicon springs is presented in this paper. Optical elements can be assembled within the tolerances of an NA=0.4 miniature microscope objective, confirming the concept of simple, zero-alignment assembly.

Nonscalar elastic light scattering from continuous random media in the Born approximation

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.

Partial-wave microscopic spectroscopy detects subwavelength refractive index fluctuations: an application to cancer diagnosis

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 diffraction-limited resolution. We show that the mesoscopic light transport theory analysis of the spectra of partial waves propagating within a weakly disordered medium, such as biological cells [i.e., partial wave spectroscopy (PWS)] quantifies refractive index fluctuations at subdiffractional length scales. We validate this nanoscale sensitivity of PWS using 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.

Association between rectal optical signatures and colonic neoplasia: potential applications for screening.

Field carcinogenesis detection represents a promising means for colorectal cancer (CRC) screening, although current techniques (e.g., flexible sigmoidoscopy) lack the requisite sensitivity. The novel optical technology low-coherence enhanced backscattering (LEBS) spectroscopy, allows identification of microscale architectural consequences of the field carcinogenesis in preclinical CRC models with unprecedented accuracy. To investigate the potential clinical translatability of this approach, we obtained biopsies from the normal-appearing rectal mucosa from patients undergoing colonoscopy (n = 219). LEBS signals were recorded through a bench-top instrument. Four parameters characterizing LEBS signal were linearly combined into a single marker. We found that LEBS signal parameters generally mirrored neoplasia progression from patients with no neoplasia, to 5 to 9 mm adenoma and to advanced adenomas. The composite LEBS marker calculated from the LEBS signal paralleled this risk status (ANOVA P < 0.001). Moreover, this was independent of CRC risk factors, benign colonic findings, or clinically unimportant lesions (diminutive adenomas, hyperplastic polyps). For advanced adenomas, the LEBS marker had a sensitivity of 100%, specificity of 80%, and area under the receiver operator characteristic curve of 0.895. Leave-one-out cross-validation and an independent data set (n = 51) supported the robustness of these findings. In conclusion, we provide the first demonstration that LEBS-detectable alterations in the endoscopically normal rectum were associated with the presence of neoplasia located elsewhere in the colon. This study provides the proof of concept that rectal LEBS analysis may potentially provide a minimally intrusive CRC screening technique. Further studies with an endoscopically compatible fiber optic probe are under way for multicenter clinical validation.

Realization of refractive microoptics through grayscale lithographic patterning of photosensitive hybrid glass.

Refractive microlenses with more than 50 microm sag are fabricated using grayscale lithography. Mechanical assembly features are made simultaneously alongside the microlenses to facilitate high precision assembly of miniature optical systems. The microlens elements are formed using lithographic patterning of photosensitive hybrid sol-gel glass requiring no etch transfer to the substrate material. Grayscale lithography enables the straightforward patterning of aspheric lenses and arbitrary surfaces within the material depth. Lessons learned in the design of a grayscale photomask are described. Characterization of the fabricated lens elements is reported including lens shape, surface quality, and image quality of a complete assembled imaging system.

Optical Detection of Buccal Epithelial Nanoarchitectural Alterations in Patients Harboring Lung Cancer: Implications for Screening

We have recently developed a novel optical technology, partial wave spectroscopic (PWS) microscopy, which is exquisitely sensitive to the nanoarchitectural manifestation of the genetic/epigenetic alterations of field carcinogenesis. Our approach was to screen for lung cancer by assessing the cheek cells based on emerging genetic/epigenetic data which suggests that the buccal epithelium is altered in lung field carcinogenesis. We performed PWS analysis from microscopically normal buccal epithelial brushings from smokers with and without lung cancer (n = 135). The PWS parameter, disorder strength of cell nanoarchitecture (L(d)), was markedly (>50%) elevated in patients harboring lung cancer compared with neoplasia-free smokers. The performance characteristic was excellent with an area under the receiver operator characteristic curve of >0.80 and was equivalent for both disease stage (early versus late) and histologies (small cell versus non-small cell lung cancers). An independent data set validated the findings with only a minimal degradation of performance characteristics. Our results offer proof of concept that buccal PWS may potentially herald a minimally intrusive prescreening test that could be integral to the success of lung cancer population screening programs.

Spectroscopic Microvascular Blood Detection From the Endoscopically Normal Colonic Mucosa: Biomarker for Neoplasia Risk

BACKGROUND & AIMS We previously used a novel biomedical optics technology, 4-dimensional elastically scattered light fingerprinting, to show that in experimental colon carcinogenesis the predysplastic epithelial microvascular blood content is increased markedly. To assess the potential clinical translatability of this putative field effect marker, we characterized the early increase in blood supply (EIBS) in human beings in vivo. METHODS We developed a novel, endoscopically compatible, polarization-gated, spectroscopic probe that was capable of measuring oxygenated and deoxygenated (Dhb) hemoglobin specifically in the mucosal microcirculation through polarization gating. Microvascular blood content was measured in 222 patients from the endoscopically normal cecum, midtransverse colon, and rectum. If a polyp was present, readings were taken from the polyp tissue along with the normal mucosa 10-cm and 30-cm proximal and distal to the lesion. RESULTS Tissue phantom studies showed that the probe had outstanding accuracy for hemoglobin determination (r(2) = 0.99). Augmentation of microvasculature blood content was most pronounced within the most superficial ( approximately 100 microm) layer and dissipated in deeper layers (ie, submucosa). EIBS was detectable within 30 cm from the lesion and the magnitude mirrored adenoma proximity. This occurred for both oxygenated hemoglobin and DHb, with the effect size being slightly greater for DHb. EIBS correlated with adenoma size and was not engendered by nonneoplastic (hyperplastic) polyps. CONCLUSIONS We show, herein, that in vivo microvascular blood content can be measured and provides an accurate marker of field carcinogenesis. This technological/biological advance has numerous potential applications in colorectal cancer screening such as improved polyp detection and risk stratification.

Modulation of light-enhancement to symbiotic algae by light-scattering in corals and evolutionary trends in bleaching.

Calcium carbonate skeletons of scleractinian corals amplify light availability to their algal symbionts by diffuse scattering, optimizing photosynthetic energy acquisition. However, the mechanism of scattering and its role in coral evolution and dissolution of algal symbioses during “bleaching” events are largely unknown. Here we show that differences in skeletal fractal architecture at nano/micro-lengthscales within 96 coral taxa result in an 8-fold variation in light-scattering and considerably alter the algal light environment. We identified a continuum of properties that fall between two extremes: (1) corals with low skeletal fractality that are efficient at transporting and redistributing light throughout the colony with low scatter but are at higher risk of bleaching and (2) corals with high skeletal fractality that are inefficient at transporting and redistributing light with high scatter and are at lower risk of bleaching. While levels of excess light derived from the coral skeleton is similar in both groups, the low-scatter corals have a higher rate of light-amplification increase when symbiont concentration is reduced during bleaching, thus creating a positive feedback-loop between symbiont concentration and light-amplification that exposes the remaining symbionts to increasingly higher light intensities. By placing our findings in an evolutionary framework, in conjunction with a novel empirical index of coral bleaching susceptibility, we find significant correlations between bleaching susceptibility and light-scattering despite rich homoplasy in both characters; suggesting that the cost of enhancing light-amplification to the algae is revealed in decreased resilience of the partnership to stress.

Measuring mucosal blood supply in vivo with a polarization-gating probe

There has been significant interest in developing depth-selective optical interrogation of biological tissue in general and of superficial (e.g., mucosal) tissue in particular. We report an in vivo polarization-gating fiber-optic probe that obtains backscattering spectroscopic measurements from a range of near-surface depths (100-200 microm). The design and testing was performed with polarized light Monte Carlo simulations and in tissue model experiments. We used the probe to investigate mucosal changes in early carcinogenesis. Measurements performed in the colonic mucosa of 125 human subjects provide the first in vivo evidence that mucosal blood supply is increased early in carcinogenesis, not only in precancerous adenomatous lesions, but also in the histologically normal-appearing tissue surrounding these lesions. This effect was primarily limited to the mucosal microcirculation and was not present in the larger blood vessels located deeper in colonic tissue.