Nanolithography and nanoscopy methods for the study of biological samples in confined spaces

Alzheimer’s, Parkinson’s and Huntington’s disease all belong to the group of amyloid pathologies also called protein misfolding diseases. Since the first discovery of amyloid fibrils of the aggregated protein tau in inclusion of Alzheimer brains samples, research has focussed on how amyloids form and their biological relevance in neurodegenerative diseases. Microfluidics are a well-established tool for the study of protein aggregation in a controllable environment, whereas super-resolution microscopy techniques have been developed and allow imaging protein misfolding in biological models in-vitro e.g. primary neural cell cultures. It was found that during the process of aggregation, misfolded proteins develop variations in toxicity which can be related to their size. Since their aggregation kinetics for different subspecies are again partially unstable depending on buffer conditions, we seek for methods for better and faster analysis of misfolding proteins on a single molecule level. Nanofluidics are capable of sampling single molecules from a concentrated solution but are not accessible to a broad community which would greatly benefit from their potential. To allow a broader community access to nanofluidic fabrication, first, a custom-built open-source two-photon lithography was implemented to demonstrate the usage of 2-photon direct laser writing for the fabrication of master molds of nanofiltration chips. The system was characterized and its nanolithography capabilities, using electron microscopy and atomic force microscopy, evaluated. The fabrication process is outlined and important details about the integration of nanofluidic functionalities into microfluidic masters elucidated. The produced chips were then used for a variety of applications ranging from single molecule diffusional measurements of Rhodamine 6G, GFP and human tau protein in solution, for entropic trapping of biological specimen and electrophoretic concentration of fluorescently labelled DNA. The flexible fabrication scheme allowed the enhancement of nanofluidic channels with adjascent nanotraps. The nanotraps allow to observe a variety of biologically relevant species such as 100 nm colloids, 40 nm colloids, exosomes, alpha-synuclein oligomers and 40 bp fluorescently labelled DNA with up to five times increased residence times in a confocal detection volume. Fluorescence burst microscopy is used to detect the residence times of the specimen with high precision. The characteristic Kramer escape time is extracted from the particles residence time probability distribution and allows an estimate of the particle size in the confined volume. The increased residence times are beneficial for photon expensive techniques to be conducted, which are hard to achieve without immobilizing them as with conventional approaches. To study biological samples related to nanofiltration and protein misfolding diseases in vitro, a custom-built pulsed-STED system was equipped with a second excitation beam path to allow for two-colour STED imaging using the same IR depletion wavelength. The system is then used on three different biological imaging applications. Single-color STED imaging is used to study the nanofiltration barrier of the kidney in minimal change disease (MCD) and compared to other super-resolution techniques such as STORM, SIM and expansion microscopy. Results show an effective alternative imaging pathway for the study of MCD without electron microscopy techniques. Secondly, STED imaging is combined with microfluidics which allows the observation and study of misfolding proteins in neural cell cultures in microfluidic devices. A microfluidic chip design consisting of two reservoirs which are connected via microchannels, allows the selective study of axonal synaptic transport processes by fluidically isolating two neural cell cultures from each other but keeping their axonal projections intact. Live neurons were exposed to human tau monomer and results indicate the inclusion of tau aggregates in lysosomes, which are then transported along the cellular microtubule network. Thirdly, STED is used to image the morphology of the endoplasmic reticulum (ER) in primary neurons. Calnexin is a chaperone-like membrane protein on the ER surface which is due to its close connection to glucoglycans of great interest to protein misfolding tauopathies. STED and expansion microcopy are demonstrated as methods to study Calnexin on the ER membrane and microtubular network with high resolution. Lastly, correlative 2-colour STED/AFM is presented on α-synuclein fibrils and synaptic vesicles, which combines fluorescence microscopy with sub-diffraction resolution and label-free mechanical mapping at the nanoscale.EPSRC CDT in Sensor Technologies for a Healthy and Sustainable Future, EPSRC CDT in Nanoscience and Nanotechnolog