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.

Cyclic-conjugated linkages between planar-macrocyclic molecules contribute to the robustness of the two-dimensional (2D) polymerization and extension of π-interactions. The fabrication of such linkages in 2D polymers remains challenging. Combining scanning tunneling microscope (STM) measurements and density functional theory (DFT) calculations, we demonstrate a linear polymerization of metal-free naphthalocyanine (NPc) molecules with [4]-radialene-like linkages on silver surfaces. Experimentally, by depositing NPc molecules on the Ag(110) surface and subsequent annealing up to 750 K, one-dimensional polymers are constructed along the [11(_)0] direction. High-resolution STM images show a stem-leaf-like feature. STM simulations based on a linear polymer of NPc molecules linked by four-membered carbon rings, [4]-radialene-like structure, agree well with the experimental observations. DFT calculations reveal that the polymerization process includes detaching two-terminal H atoms of NPc molecules along [11(_)0] direction, then bonding with a neighboring dehydrogenated NPc molecule by forming a four-membered ring. The dehydrogenation process can be promoted by on-surface impurities such as additional H atoms. Similar polymerizations have been achieved on Ag(111) surfaces in an amorphous way. Moreover, the energy gap of the NPc molecule decreases after linear polymerization, suggesting a red-shift for its optical absorption/scattering spectrum. Our study offers a new route to polymerize conjugated molecules with extended planar π-interactions.