The Ros laboratory is focused on the development and improvement of nanobiophysical techniques (in particular the combination of cutting-edge force and optical technologies) and their application to fundamental biological processes related to mechanical forces such as cell adhesion, cell and tissue mechanics and cell interactions, as well as biomolecular interactions and conformations.
Our current interests are:
Cell an tissue mechanics
Cell behavior is guided by the three-dimensional (3D) microenvironment. Reciprocal mechanical interactions between cells and their microenvironment can dictate cell phenotype and behavior, requiring studies of cells in physiologically relevant 3D extracellular matrices (ECM). In cancer development, cell and matrix stiffness has been demonstrated to be a key indicator of metastatic potential. To assess the mechanical interplay between the cells and ECM during invasion, we combined confocal fluorescence microscopy and atomic force microscopy (AFM) indentation to determine the viscoelastic properties of individual embedded cells and the pericellular matrix using novel analysis methods for heterogeneous samples.
A. Fuhrmann, J. R. Staunton, V. Nandakumar, N. Banyai, P. Davies, and R. Ros; AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells, Physical Biology 8 015007 (2011).
J. Staunton, B. Doss, S. Lindsay, R. Ros; Correlating confocal microscopy and atomic force indentation reveals metastatic cancer cells stiffen during invasion into collagen I matrices Scientific Reports 6:19686 (2016).
N. Peela, F.S. Sam; W. Christenson, D. Truong, A.W. Watson, G. Mouneimne, R. Ros, and M. Nikkhah; A Three Dimensional Micropatterned Tumor Model for Breast Cancer Cell Migration Studies, Biomaterials 81, 72-83 (2016).
P.-H. Wu, D.R.-B. Aroush, A. Asnacios, W.-C. Chen, M.E. Dokukin, B.L. Doss, P. Durand, A. Ekpenyong, J. Guck, N.V. Guz, P.A. Janmey, N.M. Moore, A. Ott, Y.-C. Poh, R. Ros, M. Sander, I. Sokolov, J.R. Staunton, N. Wang, D. Wirtz; Comparative study of cell mechanics methods, Nature Methods 15, 491-498 (2018).
H. Saini, K. Rahmani, C. Silva, M. Allam, R. Ros, M. Nikkhah; The Role of Desmoplasia and Stromal Fibroblasts on Anti-Cancer Drug Resistance in a Microengineered Tumor Model, Cellular and Molecular Bioengineering (accepted).
Non-linear super-resolution microscopy
Progress in microscopy techniques has been a key driver for new findings in biomedical sciences. Most prominently, fluorescence microscopy combines the highest sensitivity with molecular specificity and excellent image contrast. Over the last decades, the spatial resolution limit set by the diffraction of light (typically ~200 nm laterally and ~500 nm axially at a wavelength of 500 nm) has been overcome by the development and evolution of new so-called super‐resolution fluorescence microscopy methods. However, until now non-linear microscopy techniques did hardly profit yet from these super-resolution methods. Among the non-linear microscopy techniques are two-photon excitation (2PE) microscopy, second harmonic generation (SHG) imaging, and third harmonic generation (THG) imaging. We recently demonstrated the proof-of-concept for a 2PE and SHG super-resolution technique based on Image Scanning Microscopy (ISM).
I. Gregor, M. Spiecker, R. Petrovsky, J. Großhans, R. Ros, and J. Enderlein; Rapid Non‐linear Image Scanning Microscopy, Nature Methods 14, 1087–1089 (2017).
Single cell force spectroscopy is a method to quantitatively study the interaction of a single cell with surfaces or other cells. For these experiments, a single cell is attached to the cantilever of an atomic force microscope, and the interaction forces between this cell and surfaces or other cells are probed with piconewton precision.
I. Yermolenko, A. Fuhrmann, S. Magonov, V. Lishko, S. Oshkadyerov, R. Ros, and T. Ugarova; Origin of the Nonadhesive Properties of Fibrinogen Matrices Probed by Force Spectroscopy, Langmuir 26 17269–17277 (2010).
I.S. Yermolenko, O.V. Gorkun, A. Fuhrmann, V.K. Lishko, S.P. Oshkadyerov, S.T. Lord, R. Ros, and T.P. Ugarova; The assembly of nonadhesive fibrinogen matrices depends on the αC regions of the fibrinogen molecule, J. Biol. Chem. 287 41979-41990 (2012).
W. Christenson, I. Yermolenko, B. Plochberger, F. Camacho-Alanis, A. Ros, T.P. Ugarova, and R. Ros; Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices, Ultramicroscopy 136 211–215 (2014).
R. Safiullin, W. Christenson, H. Owaynat, I.S. Yermolenko, M.K. Kadirov, R. Ros and T. P. Ugarova; Fibrinogen matrix deposited on the surface of biomaterials acts as a natural anti-adhesive coating Biomaterials 67, 151-159 (2015).
Single molecule interactions
Binding forces between single receptor and ligand molecules can be investigated in dynamic force spectroscopy experiments by atomic force microscopy (AFM) or optical laser tweezers (OT) revealing details of the interaction mechanism, the kinetics of the reaction and the energy landscape of the binding. The ability to measure inter- and intramolecular forces and elasticities with pico-Newton-, nanometer- and millisecond resolution allows investigation of single ligand-receptor interaction in a broad affinity range for dissociation constants (KD) from 10-5 M to 10-15 M at a discrimination level of single point mutations. We applied this technique to a wide range of interactions including antibody-antigen, protein-DNA, protein-RNA, synthetic bioorganic interactions, supramolecular guest-host interactions, and single DNA base pairs.
R. Ros, F. Schwesinger, D. Anselmetti, M. Kubon, R. Schäfer, A. Plückthun, L. Tiefenauer; Antigen binding forces of individually addressed single-chain Fv antibody molecules: Proc. Natl. Acad. Sci. USA 95, 7402-7405 (1998).
B. Baumgarth, F. W. Bartels, D. Anselmetti, A. Becker, and R. Ros; Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA: Microbiology 151, 259-268 (2005).
A. Fuhrmann, S. Getfert, D. Anselmetti, P. Reimann, and R. Ros; Refined procedure of evaluating experimental single-molecule force spectroscopy data, Phys. Rev. E 77, 031912 (2008).
A. Fuhrmann, J.C. Schoening, D. Anselmetti, D. Staiger, and R. Ros; Quantitative analysis of single molecule RNA-protein interaction Biophysical Journal 96, 5030-5039 (2009).
Fuhrmann, S. Getfert, Q. Fu, P. Reimann, S. Lindsay, and R. Ros; Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy, Biophysical Journal 102 2381-2390 (2012).
S. Senapati, S. Biswas, S. Manna, R. Ros, S. Lindsay, P. Zhang; A Y–Shaped Three-Arm Structure for Probing Bivalent Interactions of Protein Receptor-Ligand with AFM and SPR, Langmuir , 34, 23, 6930-6940 (2018).
Progress in nanosciences and life sciences is closely related to developments of high resolution imaging technique. We are developing techniques combining atomic force microscopy with fluorescence microscopy techniques to characterize and utilize nanophotonic effects between the nanometer size AFM tip and single fluorophores.
H.G. Frey, C. Bolwien, A. Brandenburg, R. Ros, and D. Anselmetti; Optimized aperture-less optical near-field probes with 15 nm optical resolution: Nanotechnology 17, 3105–3110 (2006).
R. Eckel, V. Walhorn, Ch. Pelargus, J. Martini, J. Enderlein, Th. Nann, D. Anselmetti, and R. Ros; Fluorescence emission control of single CdSe nanocrystals using gold-modified AFM tips: Small 3, 44-49 (2007).
O. Schulz, Z. Zhao, A. Ward, M. Koenig, F. Koberling,Y. Liu, J. Enderlein, H. Yan, and R. Ros; Tip induced fluorescence quenching for nanometer optical and topographical resolution, Optical Nanoscopy 2:1 (2013)