Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells

Abstract

Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond.

Robust Visualization and Discrimination of Nanoparticles by Interferometric Imaging

“…Guided-wave evanescent excitation has several advantages over conventional TIR illumination including tighter confinement and uniformity of the excitation field over large areas. Waveguide-based dark-field illumination has been utilized for simultaneous observation of scattered and fluorescent light from multiple surface-associated nanoscopic objects using standard microscopes [21]. …”

Single-cell Analysis with Microfluidic Devices

“…Also a simple and inexpensive microfluidic flow chamber was introduced for allowing single cells to be optically trapped and analyzed. Stojanović et al 68,69 utilized surface plasmon resonance imaging for detecting and quantifying secreted antibodies from single hybridoma cells. For label-free and specific biomolecule detection at low lost, Agnarsson et al 70 invented a light-scattering microscopy technique integrated with a waveguide chip for imaging faint objects, such as sub-100 nm lipid vesicles and live cells.…”

Surface chemistry and morphology in single particle optical imaging

“…As a result of this configuration, even though the penetration depth of and evanescent field is around 100-200 nm, the depth of evanescent field can be regulated from 100 nm to a micron via tuning the thickness and refractive indices of the core and cladding layers of the waveguide [85]. With an evanescent lightscattering microscopy technique, a simultaneous fluorescent and high sensitivity scattering imaging has been done for efficient detection surface binding of BNPs in a label-free way [80]. As well as all the other techniques, in this technique, in order to obtain the highest sensitivity based on evanescent illumination, background scattering must be suppressed.…”