Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.

The solvation shell is essential for the folding and function of proteins, but how it contributes to protein misfolding and aggregation has still to be elucidated. We show that the mobility of solvation shell H 2 O molecules influences the aggregation rate of the amyloid protein α-synuclein (αSyn), a protein associated with Parkinson's disease. When the mobility of H 2 O within the solvation shell is reduced by the presence of NaCl, αSyn aggregation rate increases. Conversely, in the presence CsI the mobility of the solvation shell is increased and αSyn aggregation is reduced. Changing the solvent from H 2 O to D 2 O leads to increased aggregation rates, indicating a solvent driven effect. We show the increased aggregation rate is not directly due to a change in the structural conformations of αSyn, it is also influenced by a reduction in both the H 2 O mobility and αSyn mobility. We propose that reduced mobility of αSyn contributes to increased aggregation by promoting intermolecular interactions.

The aggregation of Aβ42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aβ42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aβ42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aβ42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aβ peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aβ peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

Temperature is a fundamental physical parameter that influences biological processes in living cells. Hence, intracellular temperature mapping can be used to derive useful information reflective of thermodynamic properties and cellular behaviour. Herein, existing publications on different thermometry systems, focusing on those that employ fluorescence‐based techniques, are reviewed. From developments based on fluorescent proteins and inorganic molecules to metal nanoclusters and fluorescent polymers, the general findings of intracellular measurements from different research groups are discussed. Furthermore, the contradiction of mitochondrial thermogenesis and nuclear‐cytoplasmic temperature differences to current thermodynamic understanding are highlighted. Lastly, intracellular thermometry is proposed as a tool to quantify the energy flow and cost associated with amyloid‐β42 (Aβ42) aggregation, a hallmark of Alzheimer's disease.

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.