A form of microscopy that utilizes a photonic crystal biosensor surface as a substrate for cell attachment enables label-free, quantitative, submicron resolution, time-resolved imaging of cell-surface interactions without cytotoxic staining agents or temporally-unstable fluorophores. Other forms of microscopy do not provide this direct measurement of live cell-surface attachment localization and strength that includes unique, dynamic morphological signatures critical to the investigation of important biological phenomena such as stem cell differentiation, chemotaxis, apoptosis, and metastasis. Here, we introduce Photonic Crystal Enhanced Microscopy (PCEM), and apply it to the study of murine dental stem cells to image the evolution of cell attachment and morphology during chemotaxis and drug-induced apoptosis. PCEM provides rich, dynamic information about the evolution of cell-surface attachment profiles over biologically relevant time-scales. Critically, this method retains the ability to monitor cell behavior with spatial resolution sufficient for observing both attachment footprints of filopodial extensions and intracellular attachment strength gradients.
Eye models are valuable tools that can help delineate the role of anatomical parameters on visual performance and guide the design of advanced ophthalmic instrumentation. We propose an optically accurate wide-field schematic eye that reproduces the complete aberration profile of the human eye across a wide visual field. The optical performance of the schematic eye is based on experimentally measured wavefront aberrations taken with a four mm pupil for the central 80°of the horizontal meridian (101 eyes) and 50°of the vertical meridian (10 eyes). Across the entire field of view, our model shows excellent agreement with the measured data both comprehensively and for low-order and high-order aberrations. In comparison to previous eye models, our schematic eye excels at reproducing the aberrations of the retinal periphery. Also unlike previous models, tilt and decentering of the gradient refractive index crystalline lens, which arose naturally through the optimization process, permits our model to mimic the asymmetries of real human eyes while remaining both anatomically and optically correct. Finally, we outline a robust reverse building eye modeling technique that is capable of predicting trends beyond those defined explicitly in the optimization routine. Our proposed model may aid in the design of wide-field imaging instrumentation, including optical coherence tomography, scanning laser ophthalmoscopy, fluorescence imaging, and fundus photography, and it has the potential to provide further insights in the study and understanding of the peripheral optics of the human eye.
Solid-state nanopore sensors are highly versatile platforms for the rapid, label-free electrical detection and analysis of single molecules, applicable to next generation DNA sequencing. The versatility of this technology allows for both large scale device integration and interfacing with biological systems. Here we report on the development of a hybrid biological solid-state nanopore platform that incorporates a highly mobile lipid bilayer on a single solid-state Al2O3 nanopore sensor, for the potential reconstitution of ion channels and biological nanopores. Such a system seeks to combine the superior electrical, thermal, and mechanical stability of Al2O3 solid-state nanopores with the chemical specificity of biological nanopores. Bilayers on Al2O3 exhibit higher diffusivity than those formed on TiO2 and SiO2 substrates, attributed to the presence of a thick hydration layer on Al2O3, a key requirement to preserving the biological functionality of reconstituted membrane proteins. Molecular dynamics simulations demonstrate that the electrostatic repulsion between the dipole of the DOPC headgroup and the positively charged Al2O3 surface may be responsible for the enhanced thickness of this hydration layer. Lipid bilayer coated Al2O3 nanopore sensors exhibit excellent electrical properties and enhanced mechanical stability (GΩ seals for over 50 h), making this technology ideal for use in ion channel electrophysiology, the screening of ion channel active drugs and future integration with biological nanopores such as α-hemolysin and MspA for rapid single molecule DNA sequencing. This technology can find broad application in bio-nanotechnology.
Abstract:The peripheral retina of the human eye offers a unique opportunity for assessment and monitoring of ocular diseases. We have developed a novel wide-field (>70°) optical coherence tomography system (WF-OCT) equipped with wavefront sensorless adaptive optics (WSAO) for enhancing the visualization of smaller (<25°) targeted regions in the peripheral retina. We iterated the WSAO algorithm at the speed of individual OCT B-scans (~20 ms) by using raw spectral interferograms to calculate the optimization metric. Our WSAO approach with a 3 mm beam diameter permitted primarily low-but also high-order peripheral wavefront correction in less than 10 seconds. In preliminary imaging studies in five normal human subjects, we quantified statistically significant changes with WSAO correction, corresponding to a 10.4% improvement in average pixel brightness (signal) and 7.0% improvement in high frequency content (resolution) when visualizing 1 mm (~3.5°) Bscans of the peripheral (>23°) retina. We demonstrated the ability of our WF-OCT system to acquire non wavefront-corrected wide-field images rapidly, which could then be used to locate regions of interest, zoom into targeted features, and visualize the same region at different time points. A pilot clinical study was conducted on seven healthy volunteers and two subjects with prodromal Alzheimer's disease which illustrated the capability to image Drusen-like pathologies as far as 32.5° from the fovea in un-averaged volume scans. This work suggests that the proposed combination of WF-OCT and WSAO may find applications in the diagnosis and treatment of ocular, and potentially neurodegenerative, diseases of the peripheral retina, including diabetes and Alzheimer's disease.
A photonic crystal (PC) surface is demonstrated as a high sensitivity platform for detection of a panel of twenty-one cancer biomarker antigens using a sandwich ELISA microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20–99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1 – 41 pg/ml), resulting in a 3-log range of quantitative detection.
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