# Label‐Free Molecular Characterization of Protein Aggregates in Differentiated Astrocytes

## TL;DR

This study uses a new imaging technique to analyze protein structures in astrocytes without labels, revealing differences between differentiated and non-differentiated cells.

## Contribution

Introduces mid-infrared photothermal imaging for label-free, multi-parameter characterization of astrocyte protein aggregates.

## Key findings

- Differentiated astrocytes show α-helical protein signatures in their processes.
- Non-differentiated astrocytes have more β-sheets and faster heat transfer.
- Photothermal imaging links protein structure with thermal resistance and function.

## Abstract

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.

Mid‐infrared photothermal microscopy enables label‐free structural, molecular, and functional imaging of protein aggregates in astrocyte cells. The processes of astrocytes differentiated on a nanomaterial interface are characterized by α‐helical signatures combined with enhanced interfacial thermal resistance properties, while the cell soma of non‐differentiated astrocytes feature richer content in β‐sheets and faster interfacial heat transfer.