Morphological Investigation of Protein Crystals by Atomic Force Microscopy

In this review, we discuss the progress in the investigation of macromolecular crystals obtained through the use of atomic force microscopy (AFM), a powerful tool for imaging surfaces and specimens at high resolution. AFM enables the visualization of soft samples at the nanoscale and can provide precise visual details over a wide size range, from the molecular level up to hundreds of micrometers. The nonperturbative nature, the ability to scan in a liquid environment, and the lack of need for freezing, fixing, or staining make AFM a well-suited tool for studying fragile samples such as macromolecular crystals. Starting from the first morphological investigations revealing the surface morphology of protein crystals, this review discusses the achievements of AFM in understanding the crystal growth processes, both at the micro- and nanoscale. The capability of AFM to investigate the sample structure at the single molecular level is analyzed considering in-depth the structure of S-layers. Lastly, high-speed atomic force microscopy (HS-AFM) is discussed as the evolution to overcome the limitations of low imaging speed, allowing for the observation of molecular dynamics and weakly adsorbed, diffusing molecules. HS-AFM has provided intuitive views and directly visualized phenomena that were previously described indirectly, answering questions that were challenging to address using other characterization methods.

Automated summary

In this review, we discuss the progress in investigating macromolecular crystals using atomic force microscopy (AFM), a powerful tool for high-resolution imaging of surfaces and specimens. AFM enables visualization of soft samples at the nanoscale, providing detailed visual details from the molecular to micrometer range. AFM is well-suited for studying fragile samples like macromolecular crystals due to its nonperturbative nature, ability to scan in liquid environments, and no need for freezing or staining. This review covers the achievements of AFM in understanding crystal growth processes at both micro- and nanoscale, analyzes its capability to investigate sample structure at the single molecular level, and discusses the evolution of high-speed AFM to overcome the limitations of low imaging speed and observe molecular dynamics.