Exploring Artifacts in Nanoscale AFM Imaging Using Quantitative Imaging Mode: a Comparison with Lipid Corrugations

Atomic Force Microscopy (AFM) enables high-resolution imaging of nanoscale structures, but the technique’s accuracy is often challenged by artifacts that resemble genuine features. This study explores how artifacts in Quantitative Imaging (QI) Mode JPK NanoWizard 4 AFM are influenced by scanning parameters, specifically Z-speed, pixel time, and scan size. By systematically adjusting these variables, we differentiated artifacts from true surface features, suggesting internal AFM limitations — particularly feedback delays and piezo scanner hysteresis — as primary contributors to artifact patterns. Our analysis revealed that changes in temporal frequency and height profiles of artifacts aligned with specific parameter variations, confirming a system-based origin. By comparing observed artifact patterns with previously documented lipid corrugation patterns, we provide insights into distinguishing genuine nanoscale features from system-induced artifacts, underscoring the importance of clear diagnostic criteria for reliable interpretation. These findings offer guidance for optimizing AFM imaging parameters, aiding in the accurate differentiation of biological features from artifacts, and enhancing the reliability of nanoscale bioimaging.