Self-Assembly of Mesoscopic Peptide Surfactant Fibrils Investigated by STORM Super-Resolution Fluorescence Microscopy

Abstract

Super-resolution fluorescence microscopy, specifically stochastic reconstruction microscopy (STORM), and atomic force microscopy (AFM) were used to image the self-assembly processes of the peptide surfactant IK. The peptide surfactants self-assembled into giant helical fibrils with diameters between 5 and 10 nm with significant helical twisting. The resolution of the STORM images was 30 nm, calculated using the Fourier ring correlation method. STORM compares favorably with AFM for the calculation of contour lengths (∼6 μm) and persistence lengths (10.1 ± 1.2 μm) due to its increased field of view (50 μm), and its ability to image bulk morphologies away from surfaces under ambient solution conditions. Two-color STORM experiments were performed to investigate the dynamic process of self-assembly after mixing of two separately labeled samples, and the results revealed the formation of long nanofibers via end-to-end connections of short ones. No evidence was found for significant monomer exchange between the samples, and the self-assembled structures were very stable and long-lived.

How should multicomponent supramolecular gels be characterised?

“…A very recent report used stochastic reconstruction microscopy (STORM) to likewise show that mixing two separately labelled samples (albeit of the same peptide) could be used to provide evidence of fibre bundling (which as noted above is very hard to resolve using TEM or AFM) to show little monomer exchange. 33 These high-resolution microscopy techniques are becoming more available, and we anticipate that there will soon be a number of papers showing their power for multicomponent systems.…”

Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

“…Here the effective point spread function (PSF) of the images is calculated based on the segmentation of the images into regions that are classified as either feature or background. Thus the peptide images were segmented into either peptide or background using Fiberapp 47 and statistical analysis of cross-sections through the diffraction limited fibres were fitted with Gaussians (the fibres are known to be 11 nm in width) 27 to provide the width of the effective point spread function i.e. the full width at half maximum minus the fibre width minus the fluorophore size, FWHM −11 nm – 1 nm.…”

“…The width distribution shifts to smaller values in the order 5 nm graphene oxide (many sheets) <1 layer of graphene oxide < no layers of graphene oxide. The width of the effective point spread function is equal to the FWHM minus 11 nm (the fibre width from AFM) 27 minus 1 nm (the size of the fluorophore). …”

“…The fibrils were aged for a week to achieve steady state structures and then labelled with Cy3B-NHS Ester fluorophores. STORM can thus be used to study the self-assembly of aggregates of the surfactant-like peptide I 3 K 27 . The quenching effect of dense packed graphene oxide layers was also studied using E. coli samples.…”

“…In our experiments a Digital sCMOS (scientific CMOS ORCA Flash 4.0 v2) camera (Hamamatsu C11440-22CU) was used to capture diffraction limited images at 100 frames per second and the rest of the STORM apparatus was as described previously 27 , 30 . A 100x objective lens (UAPON 100XOTIRF, Olympus) with a Numerical Aperture (NA) of 1.49 was used.…”