Spectral Self-interference Fluorescence Microscopy Research Articles

DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications.

Precisely Controlled Smart Polymer Scaffold for Nanoscale Manipulation of Biomolecules

We demonstrate the application of a novel smart surface to modulate the orientation of immobilized double stranded DNA (dsDNA) and the conformation of a polymer scaffold through variation in buffer pH and ionic strength. An amphoteric poly(dimethylacrylamide) based coating containing weak acrylamido acids and bases, which are copolymerized together with the neutral monomer, is covalently bound to the surface. The coating can be made to contain any desired amount of buffering and titrant ionogenic monomers, allowing control of the surface charge when the surface is bathed in a given buffer pH. Spectral self-interference fluorescence microscopy (SSFM) is utilized to precisely quantify both the DNA orientation and the polymer conformation with subnanometer resolution. It is possible to utilize the polymer scaffold to functionalize a variety of common materials used in microfabrication, making it a general purpose building block for the next generation of nanomachines and biosensors.

Closed-form representations of field components of fluorescent emitters in layered media

Dipole radiation in and near planar stratified dielectric media is studied theoretically within the context of fluorescence microscopy, as fluorescent emitters are generally modeled by electric dipoles. Although the main emphasis of this study is placed on the closed-form representations of the field components of fluorescent emitters in layered environments in near- and far-field regions, the underlying motive is to understand the limits of spectral self-interference fluorescence microscopy in studying the dipole orientation of fluorophores. Since accurate calculations of the field components of arbitrarily polarized electric dipoles in layered environments are computationally very time-consuming, a method for finding their closed-form representations is proposed using the closed-form potential Green's functions previously developed for microwave applications. The method is verified on typical geometries used in spectral self-interference microscopy experiments, where a dipole emitter is positioned over a slab of SiO(2) on top of a Si substrate. In addition to facilitating efficient calculation of near and intermediate fields of fluorescent emitters, closed-form Green's functions for fields would also play a crucial role in developing efficient and rigorous computational analysis and design tools for optical passive devices such as optical antennas by significantly improving the computational cost of the numerical solution of the integral equation.

Direct observation of conformation of a polymeric coating with implications in microarray applications.

The conformation of a three-dimensional polymeric coating (copoly(DMA-NAS-MAPS)) and immobilization and hybridization of DNA strands on the polymer coated surface are investigated. A conformational change, specifically the swelling of the surface adsorbed polymer upon hydration, is quantified in conjunction with the application of this polymer coating for DNA microarray applications. Fluorescently labeled short DNA strands (23mers) covalently linked to the functional groups on the adsorbed polymer are used as probes to measure the swelling of the polymer. A fluorescence microscopy technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), is utilized to directly measure the change in axial position of fluorophores due to swelling with subnanometer accuracy. Additionally, immobilization characteristics of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) probes, as well as hybridization of ssDNA with target strands have been studied. The results show that ssDNA further away from the surface is hybridized more efficiently, which strengthens the earlier analysis of this polymeric coating as a simple but highly efficient and robust DNA microarray surface chemistry.

Spectral Self-Interference Fluorescence Microscopy for Subcellular Imaging

Spectral self-interference fluorescence microscopy (SSFM) has been recently developed to determine the axial position of fluorescent emitters placed on reflecting dielectric structures. In this paper, we review SSFM with emphasis on its axial localization capabilities. We show that there is a tradeoff between the numerical aperture (NA) of the microscopy system and the axial localization fidelity. To use high-NA objectives for better lateral resolution, we describe and demonstrate a high-NA 4Pi microscopy system that performs spectral self-interference microscopy for axial localization of less than 2 nm. We demonstrate axial localization by using artificial samples. We also probe the membrane topography of a Shigella flexneri bacterium, several micrometers away from a solid support.

DNA conformation on surfaces measured by fluorescence self-interference

The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral self-interference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.

Spectral self-interference fluorescence microscopy

Spontaneous emission of fluorophores located close to a reflecting surface is modified by the interference between direct and reflected waves. The spectral patterns of fluorescent emission near reflecting surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra, yielding information about the location of the emitters with subnanometer precision. Spectral information was used to study thin films of fluorescent substances on surfaces.