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, critical for 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 spatial resolution can enable detailed insights into their role in brain physio-pathology. However, simultaneous insights into any structural, molecular, and functional features in unlabelled differentiated astrocytes, without perturbing their natural environment with exogenous tags, has been limited. Using mid-infrared photothermal imaging, an accumulation of α-helical signatures for 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 diffusion measurements indicate a higher interfacial thermal resistance at the astrocyte processes, connecting protein structure with thermal relaxation dynamics experimentally within the same measurement, critical for energy transport and homeostasis. This photothermal multi-parameter characterization offers unique insights into what chemically and functionally determines healthy astrocytes, paving the way towards a deeper understanding of their differentiation mechanisms. This method allows for the detection of molecular, morphological, and functional signatures associated with pathological state of astrocytes ex-vivo.