Super-resolution and single-molecule fluorescence microscopy of self-assembled peptide fibrils

Abstract

Stochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique, and single-molecule fluorescence microscopy were used to study the fibril- forming synthetic peptide I3K. STORM was extended to study self-assembled systems in 3D and slices through the fibril network were imaged in ambient solution conditions. In these experiments a lateral resolution of 30 nm was achieved, an order of magnitude improvement over diffraction limited fluorescence microscopy. Two- colour STORM experiments were also used to measure the dynamic exchange of I3K monomers between fibrils and showed that the system was very stable over a period of 4 weeks. Single-molecule fluorescence microscopy was used to measure the dynamic properties of individual I3K fibrils in the network. This showed that a significant population of fibrils were in a compressive state of pre-stress (SPS) and the distribution of fibril bending energies in the states of pre-stress followed a Levy distribution. By incorporating uncrosslinked poly(N-isopropylacrylamide) (pNIPAM) chains in the I3K network, a thermosensitive gel was created with a temperature induced change in the shear modulus of up to 3 orders of magnitude. Furthermore, single-molecule microscopy of the fibril dynamics showed that the SPS in the network were also actuated. The dynamics showed that actuation was due to the osmotic pressure exerted by the pNIPAM on the I3K network. The results presented are of significance to a range of biological structures, such as Alzheimer’s plaques and the endoplasmic reticulum, as well as therapeutic hydrogels formed from peptides like I3K. This is because the stresses shown to exist in these hydrogels will modulate the specific mechanical properties needed for their therapeutic application. Further- more, the results are of interest to polymer physicists as they provide experimental evidence of complex quenched disorder in these networks, such as states of pre-stress.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jian Lu (Supervisor) & Thomas Waigh (Supervisor)