Label‐Free Molecular Characterization of Protein Aggregates in Differentiated Astrocytes

Astrocyte cell differentiation to their characteristic starlike morphology with the expression of proteins in microdomains, criticalfor normal brain function, occurs naturally in-vivo but can be affected in pathological condition or in cell culture in-vitro.Analyzing the molecular composition and functional properties of astrocytes in a label-free manner with sub-micron spatialresolution can enable detailed insights into their role in brain physio-pathology. However, simultaneous insights into anystructural, molecular, and functional features in unlabelled differentiated astrocytes, without perturbing their natural environmentwith exogenous tags, has been limited. Using mid-infrared photothermal imaging, an accumulation of α-helical signaturesfor the extended astrocyte processes is observed in differentiated astrocytes on a nanomaterials interface. At the same time,non-differentiated astrocytes feature a more diverse protein content, rich in β-sheets. Time-resolved photothermal diffusionmeasurements indicate a higher interfacial thermal resistance at the astrocyte processes, connecting protein structure withthermal relaxation dynamics experimentally within the same measurement, critical for energy transport and homeostasis. Thisphotothermal multi-parameter characterization offers unique insights into what chemically and functionally determines healthyastrocytes, paving the way towards a deeper understanding of their differentiation mechanisms. This method allows for thedetection of molecular, morphological, and functional signatures associated with pathological state of astrocytes ex-vivo