Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells.

Single-molecule fluorescence imaging experiments generally require sub-nanomolar protein concentrations to isolate single protein molecules, which makes such experiments challenging in live cells due to high intracellular protein concentrations. Here, we show that single-molecule observations can be achieved in live cells through a drastic reduction in the observation volume using overmilled zero-mode waveguides (ZMWs- subwavelength-size holes in a metal film). Overmilling of the ZMW in a palladium film creates a nanowell of tunable size in the glass layer below the aperture, which cells can penetrate.

Palladium zero-mode waveguides for optical single-molecule detection with nanopores.

Holes in metal films do not allow the propagation of light if the wavelength is much larger than the hole diameter, establishing such nanopores as so-called zero-mode waveguides (ZMWs). Molecules, on the other hand, can still pass through these holes. We use this to detect individual fluorophore-labelled molecules as they travel through a ZMW and thereby traverse from the dark region to the illuminated side, upon which they emit fluorescent light.

Cytoplasmic flows in starfish oocytes are fully determined by cortical contractions.

Cytoplasmic flows are an ubiquitous feature of biological systems, in particular in large cells, such as oocytes and eggs in early animal development. Here we show that cytoplasmic flows in starfish oocytes, which can be imaged well with transmission light microscopy, are fully determined by the cortical dynamics during surface contraction waves. We first show that the dynamics of the oocyte surface is highly symmetric around the animal-vegetal axis.

Backbone circularization of Bacillus subtilis family 11 xylanase increases its thermostability and its resistance against aggregation.

The activity of proteins is dictated by their three-dimensional structure, the native state, and is influenced by their ability to remain in or return to the folded native state under physiological conditions. Backbone circularization is thought to increase protein stability by decreasing the conformational entropy in the unfolded state. A positive effect of circularization on stability has been shown for several proteins.