Micron 48 (2013) 26–33 Contents lists available at SciVerse ScienceDirect Micron j o ur nal homep age: www.elsevier.com/locate/micron Imaging living cells surface and quantifying its properties at high resolution using AFM in QI TM mode L. Chopinet a,b,c,1 , C. Formosa a,c,d,e,1 , M.P. Rols b,c , R.E. Duval e,f,g , E. Dague a,c,d,∗ a Centre National de la Recherche Scientifique, Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Toulouse, France b Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France c Université de Toulouse, Toulouse, France d Institut des Technologies Avancées en Sciences du Vivant, Toulouse, France e CNRS, SRSMC (Structure et Réactivité des Systèmes Moléculaires Complexes), UMR 7565, Nancy, France f Université de Lorraine, SRSMC, UMR 7565, Faculté de Pharmacie, Nancy, France g ABC PlatformR, Nancy, France a r t i c l e i n f o Article history: Received 13 December 2012 Received in revised form 6 February 2013 Accepted 7 February 2013 Keywords: Atomic force microscopy Quantitative imaging Microorganisms Eukaryotic cells Imaging Nanomechanical properties a b s t r a c t Since the last 10 years, AFM has become a powerful tool to study biological samples. However, the classical modes offered (imaging or tapping mode) often damage sample that are too soft or loosely immobilized. If imaging and mechanical properties are required, it requests long recording time as two different experiments must be conducted independently. In this study we compare the new QI TM mode against contact imaging mode and force volume mode, and we point out its benefit in the new challenges in biology on six different models: Escherichia coli, Candida albicans, Aspergillus fumigatus, Chinese hamster ovary cells and their isolated nuclei, and human colorectal tumor cells. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Since 25 years, Atomic Force Microscopy has emerged as a valu- able tool in biology, to study the morphology of living cells, their surface roughness, and theirs nanomechanical properties (elastic- ity through Young modulus (YM) values, Single molecule force spectroscopy) (Müller and Dufrêne, 2011). Technological improve- ments were required to make this jump from physics to biology. Classically, AFM provides two imaging modes to probe biolog- ical sample known as contact mode and tapping mode. In contact mode, the AFM tip raster scans over the sample to obtain high res- olution images of sample surface in terms of height, the sample topography being measured by detecting changes in the deflec- tion of the tip as a function of position on the surface (Liu and Wang, 2010). However, when applied to deformable soft samples, the resulting topographic images are poorly correlated with the variations in height across the sample since the AFM tip deforms ∗ Corresponding author at: Centre National de la Recherche Scientifique, Labora- toire d’Analyse et d’Architecture des Systèmes (LAAS), 7 avenue du colonel Roche, F-31400 Toulouse, France. Tel.: +33 561337841. E-mail address: edague@laas.fr (E. Dague). 1 These two authors contributed equally to the work. the surface during the raster scan. As a second imaging mode, taping mode allows to image soft sample and with a very good resolution (Milhiet et al., 2011). In this mode, very stiff cantilever is used, and is oscillating near its resonance frequency during the scan, with- out being in contact with the sample. Change in the amplitude of oscillation during raster scanning report on the surface topog- raphy. Consequently, the lateral forces between the tip and the sample can be significantly reduced, which, in principle, avoid the deformation artifacts associated with contact-mode imaging. How- ever, in a biological system where the electrolyte concentration is high, interactions with low-range surface forces affect the vibrating tip during its trajectory. These forces can influence the oscillation amplitude; therefore the contact between the tip and the sample becomes unavoidable, leading to a deformation of the sample. Recent developments have conducted to high speed AFM (Kodera et al., 2010) or multi-frequency force spectroscopy (Garcia and Herruzo, 2012) in order to image sample faster. Those two modes overcome the time limitation by increasing the scan rate. However no biophysical properties can be extracted from the data, since these two advanced modes functions with oscillating tips. Indeed, besides topography imaging, AFM can also be used in force spectroscopy mode to measure biophysical properties of samples (such as elasticity and molecular organization of the sample sur- face) (Formosa et al., 2012b; Heinisch and Dufrene, 2010). A major 0968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micron.2013.02.003