Biology subjects

Kirichuk, O.

Publications and source records attributed to Kirichuk, O..

2 recordsLinked to original sources

bioRxiv2024

Optical sectioning for reflection interference microscopy

Reflection Interference Contrast Microscopy (RICM, also known as interference reflection microscopy) and related techniques have become of wide interest to the biophysical, soft matter and biochemistry communities owing to their exquisite sensitivity for characterising thin films or individual nanoscopic objects adsorbed onto surfaces, or for monitoring cell-substrate interactions. Over the recent years, striking progresses have been made to improve the sensitivity and the quantitative analysis of RICM. Its use in more complex environments, with spurious reflections stemming from a variety of structures in the sample, remains however challenging. In this paper, we demonstrate two optical sectioning methods that effectively reduce such background and can be readily implemented in a conventional RICM setup: line confocal detection, and structured illumination microscopy. We characterise experimentally the benefits to image quality and demonstrate the use of the methods for quantitative imaging of complex biological and biomimetic samples: cellular membranes, thin organic films, surface biofunctionalization. We then discuss the benefits of each method and provide guidelines to arbitrate between sectioning and signal-to-noise ratio. Finally, we provide a detailed description of our experimental setup and a home-written image acquisition and processing software that should allow the interested reader to duplicate such a setup on a home-built or commercial microscope.

biophysics↗

bioRxiv2023

Competitive specific anchorage of molecules onto surfaces: quantitative control of grafting densities and contamination by free anchors

The formation of surfaces decorated with biomacromolecules such as proteins, glycans or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes, and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest, and present effective guidelines towards optimizing incubation conditions for the quantitative control of surface densities. Focussing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

biophysics↗