Optimizing Single‐Cell Measurement Using Dynamic Atomic Force Microscopy

Advancements in atomic force microscopy (AFM) have established it as a versatile tool for imaging and characterizing biological samples, including living single cells in physiological conditions. Utilizing higher eigenmodes and harmonics of AFM microcantilevers offers enhanced sensitivity for measuring cells, tissues, and microorganisms. However, achieving accurate measurements in liquid environments poses challenges, particularly in detecting the cantilever's response. Optimizing the optical beam detection system is crucial to improving measurement sensitivity and image quality, especially when using higher‐order vibration modes. This study addresses these challenges by optimizing the optical detection sensitivity of microcantilevers in dynamic and high‐frequency AFM for single‐cell measurements. This study investigates the effect of laser positioning on the cantilever's dynamic response in liquid environments and its impact on imaging resolution. Advanced image analysis techniques, such as natural image quality evaluator, cross‐correlation, and sharpness, are employed to evaluate image quality. This approach provides insights and general guidelines for obtaining high‐resolution images as well as image analysis approaches of biological materials in physiological conditions.