Scanning Probe Microscopy Characterization of Biomolecules enabled by Mass‐Selective, Soft‐landing Electrospray Ion Beam Deposition

Abstract

Scanning probe microscopy (SPM), in particular at low temperature (LT) under ultra‐high vacuum (UHV) conditions, offers the possibility of real‐space imaging with resolution reaching the atomic level. However, its potential for the analysis of complex biological molecules has been hampered by requirements imposed by sample preparation. Transferring molecules onto surfaces in UHV is typically accomplished by thermal sublimation in vacuum. This approach however is limited by the thermal stability of the molecules, i.e. not possible for biological molecules with low vapour pressure. Bypassing this limitation, electrospray ionisation offers an alternative method to transfer molecules from solution to the gas‐phase as intact molecular ions. In soft‐landing electrospray ion beam deposition (ESIBD), these molecular ions are subsequently mass‐selected and gently landed on surfaces which permits large and thermally fragile molecules to be analyzed by LT‐UHV SPM. In this concept, we discuss how ESIBD+SPM prepares samples of complex biological molecules at a surface, offering controls of the molecular structural integrity, three‐dimensional shape, and purity. These achievements unlock the analytical potential of SPM which is showcased by imaging proteins, peptides, DNA, glycans, and conjugates of these molecules, revealing details of their connectivity, conformation, and interaction that could not be accessed by any other technique.