title: Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.
abstract: A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers.
authors.affiliation: CEA, BIAM, LBDP, F-13108 Saint Paul lez Durance, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Godon
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France; CNRS, IBS, F-38044 Grenoble, France; CEA, IBS, F-38044 Grenoble, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Teulon
authors.affiliation: ICSM-UMR5257 CEA/CNRS/UM2/ENSCM, F-30207 Bagnols sur Cèze, France.
authors.forename: Michael
authors.initials: M
authors.lastname: Odorico
authors.affiliation: CEA, BIG, F-38054 Grenoble, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Basset
authors.affiliation: Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France; CNRS, ISM, UMR 5255, F-33400 Talence, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Meillan
authors.affiliation: Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France; CNRS, ISM, UMR 5255, F-33400 Talence, France.
authors.forename: Luc
authors.initials: L
authors.lastname: Vellutini
authors.affiliation: 478 rue Cyprien Jullin, 38470 Vinay, France.
authors.forename: Shu-Wen W
authors.initials: SW
authors.lastname: Chen
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France; CNRS, IBS, F-38044 Grenoble, France; CEA, IBS, F-38044 Grenoble, France. Electronic address: jlpellequer@cea.fr.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Pellequer
mesh_terms: D018625:Microscopy, Atomic Force / Q000379:methods*; D014027:Tobacco Mosaic Virus / Q000648:ultrastructure
keywords: AFM topography; Mica; Peakforce tapping; Self-assembled monolayers; Single molecule deformation; Tobacco mosaic virus