Oblique-incidence Reflectivity Difference Microscope Research Articles

Detection speed optimization of the OI-RD microscope for ultra-high throughput screening.

The oblique-incidence reflectivity difference (OI-RD) microscope is a label-free detection system for microarrays that has many successful applications in high throughput drug screening. The increase and optimization of the detection speed of the OI-RD microscope will enable it to be a potential ultra-high throughput screening tool. This work presents a series of optimization methods that can significantly reduce the time to scan an OI-RD image. The wait time for the lock-in amplifier was decreased by the proper selection of the time constant and development of a new electronic amplifier. In addition, the time for the software to acquire data and for translation stage movement was also minimized. As a result, the detection speed of the OI-RD microscope is 10 times faster than before, making the OI-RD microscope suitable for ultra-high throughput screening applications.

Label-Free Sensing on Microarrays.

Microarrays of biological molecules such as DNAs, proteins, carbohydrates, and small molecules provide a high-throughput platform for screening tens of thousands of biomolecular interactions simultaneously, facilitating the functional characterization of these biomolecules in areas of genomics, proteomics, glycomics, and cytomics. Routinely, analysis of binding reactions between solution-phased probes and surface-immobilized targets involves some kinds of fluorescence-based detection methods. Even though these methods have advantages of high sensitivity and wide dynamic range, labeling probes and/or targets inevitably changes their innate properties and in turn affects probe-target interactions in often uncharacterized ways. Therefore, in recent years, various label-free sensing technologies have been developed for characterizing biomolecular interactions in microarray format. These biosensors, to a certain extent, take the place of fluorescent methods by providing a comparable sensitivity as well as retaining the conformational and functional integrality of biomolecules to be investigated. More importantly, some of these biosensors are capable of real-time monitoring probe-target interactions, providing the binding affinities of these reactions. Using label-free biosensors in microarrays has become a current trend in developing high-throughput screening platforms for drug discoveries and applications in all areas of "-omics." This article is aimed to provide principles and recent developments in label-free sensing technologies applicable to microarrays, with special attentions being paid to surface plasmon resonance microscopy and oblique-incidence reflectivity difference microscopy.

An ellipsometry-based biosensor for label-free, real-time, and in-situ detection of DNA-DNA and DNA-protein interactions

An ellipsometry-based biosensor, the oblique-incidence reflectivity difference (OI-RD) scanning microscope, was developed for label-free, real-time, and in-situ detection of biomolecular interactions. We successfully demonstrate that DNA-DNA hybridization and DNA-protein reactions in a DNA microarray can be monitored by such an OI-RD microscope.

AN OBLIQUE-INCIDENCE REFLECTIVITY DIFFERENCE STUDY OF THE DEPENDENCE OF PROBE-TARGET REACTION CONSTANTS ON SURFACE TARGET DENSITY USING STREPTAVIDIN-BIOTIN REACTIONS AS A MODEL

A combination of the microarray platform and oblique-incidence reflectivity difference (OI-RD) microscopy was used to study the dependence of kinetic constants of probe-target reactions on the surface density of immobilized targets. Streptavidin-biotin reactions with a very high binding affinity were employed as the study model. Oblique-incidence reflectivity difference microscopy, a label-free and surface-based detection technique, was developed for monitoring real-time binding curves between two interactive biomolecules, enabling the acquisition of kinetic constants such as on-rate, off-rate, and equilibrium dissociation constants. These kinetic constants are important in characterizing biomolecular interactions because in living cells all intercellular and intermolecular reactions are at dynamic rather than at stable equilibrium. The kinetic constant of streptavidin binding to surface-immobilized biotin-bovine serum albumin was demonstrated to be significantly affected by the density of surface bovine serum albumin conjugates, mainly due to mass-transport effects within targets.

Use of real-time, label-free analysis in revealing low-affinity binding to blood group antigens by Helicobacter pylori.

Infectious diseases are often initiated by microbial adherence that is mediated by the binding of attachment molecules, termed adhesins, to cell surface receptors on host cells. We present an experimental system, oblique-incidence reflectivity difference (OI-RD) microscopy, which allows the detection of novel, low-affinity microbial attachment mechanisms that may be essential for infectious processes. OI-RD microscopy was used to analyze direct binding of the oncopathogen, Helicobacter pylori ( H. pylori ) to immobilized glycoconjugates in real time with no need for labeling tags. The results suggest the presence of additional Lewis b blood group antigen (Le(b)) binding adhesins that have not been detected previously. OI-RD microscopy also confirmed the high-affinity binding of H. pylori outer-membrane protein BabA to Le(b). The OI-RD microscopy method is broadly applicable to real-time characterization of intact microbial binding to host receptors and offers new strategies to elucidate the molecular interactions of infectious agents with human host cells.

Label-free Detection of Surface Markers on Stem Cells by Oblique-Incidence Reflectivity Difference Microscopy

Conventional fluorescence microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in often uncharacterized ways. Here we present a method using an oblique-incidence reflectivity difference (OI-RD) microscope for label-free, real-time detection of cell surface markers, and apply it to analysis of stage-specific embryonic antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces, and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies bind only to the surface of the stem cells and not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. Thus, the OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells; this information could be useful for determination of stem cell stages.

Reflecting on Stem Cell Differentiation

Reflecting on Stem Cell Differentiation

Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format

We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in. x 3 in. (2.54 cm x 7.62 cm) microscope slide.

Label-free detection of microarrays of biomolecules by oblique-incidence reflectivity difference microscopy.

We developed an oblique-incidence reflectivity difference (OI-RD) scanning microscope for label-free imaging of microarrays of biomolecules upon solid substrates. We demonstrate that hybridization reactions in an oligonucleotide microarray fabricated upon a glass slide can be detected by such an OI-RD microscope.