Advances in Scanning Probe Microscopy for Biological Systems

Scanning Probe Microscopy (SPM), as a nanoscale characterization technique, employs a sharp probe to detect local tip-sample interactions through near-field physical phenomena. This approach achieves atomic-resolution surface imaging while enabling concurrent characterization of multi-parametric properties—electrical, magnetic, and chemical signals. This review offers a cross-disciplinary perspective on the advances in SPM for biological systems, which serves as a practical guide for life scientists to select from the expanding array of SPM techniques. We outline the fundamental principles of Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM), before discussing a series of advanced SPM techniques: force spectroscopy for nanomechanical characterization, Kelvin Probe Force Microscopy (KPFM) for surface potential imaging, Scanning Near-field Optical Microscopy (SNOM) for super-resolution optics, Tip-Enhanced Raman Spectroscopy (TERS) for nanoscale chemical identification, and Scanning Electrochemical Microscopy (SECM) for localized electrochemical activity detection. A systematic comparison of these technologies provides researchers with clear criteria to select the optimal methodology for diverse demands, either characterizing nucleic acids and proteins or analyzing single-cell ultrastructure and biomechanics. In addition, this review explores the transformative integration of SPM and Artificial Intelligence (AI). This integration is expected to automate SPM workflows. It will also increase the stability of SPM systems and enhance the reproducibility of experimental results. Furthermore, by addressing current challenges and future perspectives of in vivo imaging, this review aims not merely to review the progress but to empower biologists to harness these intelligent multi-modal SPM systems for groundbreaking discoveries.