The hyperspectral, interferometric microscopy technique, PWS has demonstrated the ability to measure variance in the nanoscale refractive index (Σ) of chromatin – the macromolecular assembly containing most of a cell’s genetic material. However, the question arises: how does Σ relate to the physical distribution of mass in chromatin, and specifically the organization of chromatin packing. We developed an analytical framework to relate Σ to the mass-density autocorrelation function – which can fully describe the distribution of mass and is characterized by D. This relationship was validated numerically using the rigorous modelling technique FDTD and experimentally with PWS and Chromatin Electron Microscopy (ChromEM).
While there are a plethora of in vivo fiber-optic spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials with highly trained personnel familiar with the operation of experimental optical technologies, very few techniques show the same level of success in large multicenter trials. To meet the stringent requirements for a viable optical spectroscopy system to be used in a clinical setting, we developed components including an automated calibration tool, optical contact sensor for signal acquisition, and a methodology for real-time in vivo probe calibration correction. The end result is a state-of-the-art medical device that can be realistically used by a physician with spectroscopic fiber-optic probes. We show how the features of this system allow it to have excellent stability measuring two scattering phantoms in a clinical setting by clinical staff with ∼0.5 % standard deviation over 25 unique measurements on different days. In addition, we show the systems’ ability to overcome many technical obstacles that spectroscopy applications often face such as speckle noise and user variability. While this system has been designed and optimized for our specific application, the system and design concepts are applicable to most in vivo fiber-optic-based spectroscopic techniques.
Fluorescence photo-switching of native, unmodified DNA using visible light enables label-free, nanoscale, single-molecule photon localization microscopy (PLM) of chromatin structure. Compared with conventional label-based super resolution imaging techniques, the label-free DNA-PLM has the advantage of faithfully resolving the native nucleotides under non-perturbing conditions, thus allowing a reliable analysis of the chromatin organization. Recently, we have developed an algorithm to quantify the chromatin spatial distribution based on label-free DNA-PLM images by calculating the fractal dimension from the chromatin cluster size and the number of photon emission events. For demonstration, we employed label-free DNA-PLM with TIRF illumination, and imaged the nuclei of ovarian cancer cells with three descending chromatin heterogeneities: the P53 mutation (M248), the wild type (A2780), and the wild type treated with a commonly-used chemotherapeutic drug celecoxib (Cele). Using the algorithm, we extracted the fractal dimensions for nuclear chromatin. We found that the fractal dimension is between 2 to 3 for all cells, which lies in the range of reported values from other techniques (e.g., TEM). We also observed that M248 has the highest fractal dimension while Cele has the lowest, a perfect match with the experimental expectations. We believe this study can provide a new approach to quantify label-free super-resolution imaging of macromolecular structures and could contribute to our knowledge of native in-vitro nuclear chromatin configurations.
We demonstrate a multimodal imaging methodology to probe the nanoscale environment of cells. The system combines partial-wave spectroscopic (PWS) microscopy and spectroscopic photon localization microscopy (SPLM). PWS quantifies the nanoarchitecture of cells with sensitivity to structures between 20 and 200 nm. SPLM is a newly developed super-resolution imaging technique based upon the principles of single-molecule localization microscopy and spectroscopy. In addition to allowing super-resolution imaging, SPLM provides inherent molecular-specific spectroscopic information of targeted structures visualized when dyes are used. Combining both of these modalities into a single instrument allows nanoscale characterization of the super-resolution molecular imaging provided by SPLM as it relates to nanoscale structural information provided by PWS. As an example, we labeled RNA polymerase in HeLa cells and showed correlations between the locations of the RNA polymerase visualized by SPLM and the nanoscale structure of the chromatin measured by PWS. Such information is crucial in understanding the role of specific molecules in regulating the chromatin structure and gene expression. More broadly, this instrument can give insight into the molecular pathways of diseases and therapeutic treatments of those diseases, while simultaneously showing the effects on chromatin topology.
While there are a plethora of in-vivo spectroscopic techniques that have demonstrated the ability to detect a number of diseases in research trials, very few techniques have successfully become a fully realized clinical technology. This is primarily due to the stringent demands on a clinical device for widespread implementation. Some of these demands include: simple operation requiring minimal or no training, safe for in-vivo patient use, no disruption to normal clinic workflow, tracking of system performance, warning for measurement abnormality, and meeting all FDA guidelines for medical use. Previously, our group developed a fiber optic probe-based optical sensing technique known as low-coherence enhanced backscattering spectroscopy (LEBS) to quantify tissue ultrastructure in-vivo. Now we have developed this technique for the application of prescreening patients for colonoscopy in a primary care (PC) clinical setting. To meet the stringent requirements for a viable medical device used in a PC clinical setting, we developed several novel components including an automated calibration tool, optical contact sensor for signal acquisition, and a contamination sensor to identify measurements which have been affected by debris. The end result is a state-of-the-art medical device that can be realistically used by a PC physician to assess a person’s risk for harboring colorectal precancerous lesions. The pilot study of this system shows great promise with excellent stability and accuracy in identifying high-risk patients. While this system has been designed and optimized for our specific application, the system and design concepts are universal to most in-vivo fiber optic based spectroscopic techniques.
We present an ultra-simple miniature fiber optic probe to measure spatially and spectrally resolved diffuse reflectance in the sub-diffuse regime (i.e. measurements with source-detector separation less than a transport mean free path) in-vivo. This probe has a robust and simple design with a small footprint (<.5 mm diameter). We show that our probe has sensitivity to structures scattering light an order of magnitude smaller than the diffraction limit, and thus can be used to quantify alterations in the very smallest structures in tissue (e.g. organelles, chromatin, collagen fibers, etc.). Specifically, the probe samples the spatial profile of diffuse reflectance in the sub-diffusion regime (P(r), r<<1 mm). P(r) can be used to quantify the entire shape of the phase function, F(θ). The shape of the refractive index correlation function Bn(rd) (through which the spatial distribution of mass is defined) can be analytically derived from the shape of F(θ) through application of the Born approximation. Therefor measurements of P(r) can elucidate F(θ) and Bn(rd). This ability has tremendous potential for use as a diagnostic tool and broad applications for probing the nanoscale environment of tissue in-vivo.
Diverging beam illumination is widely used in many optical techniques especially in fiber optic applications and coherence phenomenon is one of the most important properties to consider for these applications. Until now, people have used Monte Carlo simulations to study the backscattering coherence phenomenon in collimated beam illumination only. We are the first one to study the coherence phenomenon under the exact diverging beam geometry by taking into account the impossibility of the existence for the exact time-reversed path pairs of photons, which is the main contribution to the backscattering coherence pattern in collimated beam. In this work, we present a Monte Carlo simulation that considers the influence of the illumination numerical aperture. The simulation tracks the electric field for the unique paths of forward path and reverse path in time-reversed pairs of photons as well as the same path shared by them. With this approach, we can model the coherence pattern formed between the pairs by considering their phase difference at the collection plane directly. To validate this model, we use the Low-coherence Enhanced Backscattering Spectroscopy, one of the instruments looking at the coherence pattern using diverging beam illumination, as the benchmark to compare with. In the end, we show how this diverging configuration would significantly change the coherent pattern under coherent light source and incoherent light source. This Monte Carlo model we developed can be used to study the backscattering phenomenon in both coherence and non-coherence situation with both collimated beam and diverging beam setups.
Reflectance measurements acquired from within the subdiffusion regime (i.e., lengthscales smaller than a transport mean free path) retain much of the original information about the shape of the scattering phase function. Given this sensitivity, many models of subdiffusion regime light propagation have focused on parametrizing the optical signal through various optical and empirical parameters. We argue, however, that a more useful and universal way to characterize such measurements is to focus instead on the fundamental physical properties, which give rise to the optical signal. This work presents the methodologies that used to model and extract tissue ultrastructural and microvascular properties from spatially resolved subdiffusion reflectance spectroscopy measurements. We demonstrate this approach using ex-vivo rat tissue samples measured by enhanced backscattering spectroscopy.
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