Super-High-Resolution Densitometry by Optical Trapping of a Single-Molecularly Imprinted Polymer Particle for Subsingle Molecule Detection.

In this study, for the first time, we have shown that the single nano-/microparticle trapping ability of optical tweezers combined with the high selectivity of molecularly imprinted polymers (MIPs) provides an indispensable molecular-level instrument for chemical sciences. Trapping a single MIP inside a solution and analyzing its Brownian motion allow for real-time determination of its target molecule [trimipramine (TMP) in our case] content. This method is also utilized to precisely measure TMP concentration in the bulk solution. The detection and optical volumes, respectively, defined as single MIP volume and laser focal volume, were about a few femtoliters. According to our data within a detection volume inside the bulk solution, 0.02-0.25 target molecules could be detectable with a detection limit of 0.005 molecules. Thus, we detected 1/1000th of the subsingle molecule in detection volume by high-resolution densitometry.

Fluorescent quantification of size and lamellarity of membrane nanotubes.

Membrane nanotubes, ubiquitous in cellular systems, adopt a spectrum of curvatures and shapes that are dictated by their intrinsic physical characteristics as well as their interactions with the local cellular environment. A high bending flexibility is needed in the crowded cytoplasm where tubes often need to bend significantly in the axial direction at sub-micron length scales. We find the stiffness of spontaneously formed membrane nanotubes by measuring the persistence length of reconstituted membrane nanotubes freely suspended in solution and imaged by fluorescence microscopy. By quantifying the tube diameter we demonstrate for the first time that the persistence length scales linearly with radius. Although most tubes are uni-lamellar, the predicted linear scaling between tube radius and persistence length allows us to identify tubes that spontaneously form as multilamellar structures upon hydration. We provide the first experimental evidence that illumination of lipid fluorophores can have a profound effect on the lipid bilayer which we sensitively detect as a continuous change in the tube persistence length with time. The novel assay and methodology here presented has potential for quantification of the structural reinforcement of membrane tubes by scaffolding proteins.