Wide-field interferometric microscopy is a common-path interferometry technique that allows for label-free and high-throughput detection of weakly scattering sub-diffraction-limited biological nanoparticles. Such nanoparticles appear as diffraction-limited-spots in the image and optically resolving them beyond their ‘digital’ detection still remains a challenge owing to the diffraction barrier as well as the typical signal levels that fall below the noise floor. In this study, we demonstrate the utility of computational optics in the interference enhanced nanoparticle imaging to improve its resolving power to obtain structural information on clinically relevant and often complexed-shaped biological nanoparticles such as viruses and exosomes. We consider a spatially incoherent structured illumination based image reconstruction strategy in wide-field interferometric microscopy to achieve high contrast nanoparticle imaging with super-resolution. Our reconstruction technique makes use of the optical transfer function of the system derived via an analytical model based on angular spectrum representation. We provide experimental demonstrations using an artificial sample to quantify the resolution enhancement as well as a biological sample for concept demonstration. We also benchmark the results against gold standard images obtained using an electron microscope. Our highly-sensitive super-resolution imaging system constitutes a noncomplex optical design, which can be realized with simple modifications to a conventional epi-illumination microscope, offering a cost-effective alternative to the laborious and expensive standard high-resolution microscopy techniques. It has a broad spectrum of applications ranging from clinical diagnostics to biotechnological research.
The standard laboratory procedure for determining the antibiotic susceptibility of a pathogen (an antimicrobial susceptibility test, AST) measures the inhibition of growth, and requires several days. This can delay effective therapy and lead to antibiotic overuse and misuse. Recent work (Wei Hou et al, Lab on a Chip 2015) has shown that resistant and susceptible pathogens will have very different gene expression profiles shortly following antibiotic exposure, and that these expression biomarkers may be used to accurately identify the pathogen species, strain and antibiotic susceptibility without growth. We therefore developed an ultrasensitive ‘digital microarray’ for performing rapid & quantitative gene expression analysis as part of a rapid AST. The digital microarray uses plasmonic gold nanorods (GNRs) functionalized with DNA to specifically label each target RNA that binds to the microarray. Each GNR on the array is then individually detected based on its light scattering, with an interferometric microscopy technique called SP- IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Due to its single molecule readout, the assay has a limit of detection of less than 1 femtomolar following just 2 hours of incubation. Altogether, digital microarrays are about 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing. The reproducibility and robustness of the multiplexed assay will next be evaluated with clinically relevant pathogenic strains of E. coli as part of a functional rapid AST.
We have demonstrated Interferometric Reflectance Imaging Sensor (IRIS) with the ability to detect single nanoscale particles. By extending single-particle IRIS to in-liquid dynamic imaging, we demonstrated real-time digital detection of individual viral pathogens as well as single molecules labeled with Au nanoparticles. With this technique we demonstrate real-time simultaneous detection of multiple targets in a single sample, as well as quantitative dynamic detection of individual biomolecular interactions for reaction kinetics measurements. This approach promises to simplify and reduce the cost of rapid diagnostics.
In this paper, we demonstrate utilization of a commercial flatbed document scanner as a label-free biosensor for highthroughput imaging of DNA and protein microarrays. We implemented an interferometric sensing technique through use of a silicon/oxide layered substrate, and easy to implement hardware modifications such as re-aligning moving parts and inserting a custom made sample plate. With a cost as low as 100USD, powered by a USB cable, and scan speed of 30 seconds for a 4mm x 4 mm area with ~10μm lateral resolution, the presented system offers a super low cost, easy to use alternative to commercially available label-free systems.
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