Pushing the limits of atomic force microscopy: Emerging methods for speed, resolution, and quantitative analysis.

Since its introduction 40 years ago, atomic force microscopy (AFM) has transformed nanoscale imaging across the physical and life sciences. In biology, AFM has provided unprecedented insights into nanoscale biomolecular organisation under near-physiological conditions, revealing structures and interactions inaccessible to many conventional approaches. However, biological function is inherently dynamic, and understanding molecular mechanisms requires moving beyond static structural snapshots towards direct observation of processes across multiple spatial and temporal scales. In this review, we discuss emerging approaches that are extending the capabilities of AFM beyond traditional imaging, pushing the limits of temporal resolution, spatial precision, quantitative measurement, and data analysis. We highlight advances in speed that enable direct visualisation of molecular dynamics, approaches for sub-millisecond measurements, methods that overcome conventional resolution limits, and computational approaches that enhance image analysis, automation, and information extraction from complex datasets. Together, these developments are transforming AFM from a technique focused primarily on high-resolution structural imaging into a platform for dynamic, quantitative, and high-throughput investigation of biological systems. Finally, we consider how these advances may shape the next 40 years of AFM, enable autonomous measurements and reveal biological mechanisms across previously inaccessible spatial and temporal scales.