Bioanalytical TERS and Advanced Data Processing Methods

ABSTRACT Tip‐enhanced Raman spectroscopy (TERS) enables vibrational imaging with subnanometer spatial resolution, offering label‐free chemical insight into biological structures at the single‐molecule level. This review outlines recent developments in TERS instrumentation, including tip fabrication strategies, excitation geometries, and configurations compatible with ambient and liquid‐phase environments. Emphasis is placed on biological applications, where TERS has been used to probe proteins, nucleic acids, lipid assemblies, and viral particles. These studies demonstrate how localized plasmonic enhancement supports surface‐selective detection and enables mapping of molecular heterogeneity across cellular interfaces and membrane domains. Spatial resolution metrics, both lateral and vertical, are discussed in relation to tip–sample coupling, field confinement, and operational conditions. We also examine methodological challenges such as signal instability, tip degradation, thermal effects, and reproducibility, alongside emerging strategies for mitigation. In parallel, we highlight the role of advanced data analysis frameworks, including multivariate chemometric methods and machine learning approaches, which are increasingly essential for interpreting hyperspectral datasets and extracting relevant insights. Together, these advances position TERS as a versatile platform for nanoscale bioanalysis, with growing relevance for mechanistic studies, molecular diagnostics, and real‐time tracking of biochemical processes.