[Zurück]

R. Zhu, M. Duman, J. Madl, G. Schütz, P. Hinterdorfer:
"Combined Topography, Recognition, and Fluorescence Measurements on Cells";
in: "Cell Membrane Nanodomains - from Biochemistry to Nanoscopy, 
editors: Alessandra Cambi and Diane S. Lidke9", CRC Press, Taylor & Francis Group, 2014, ISBN: 978-1-4822-0991-4, S. 367 - 390.

16.1 COMBINED OPTICAL AND ATOMIC FORCE MICROSCOPE
Conventional optical microscopy techniques, such as bright field, cross-polarized light, phase contrast, dark field, and differential interference contrast provide morphological and structural information of cells and cellular organelles, while fluorescence microscopy allows for imaging specific molecular components and for determining the localization of molecules in cells down to the single-molecule level,1 making it possible to follow cellular processes and to monitor the dynamics of living cell components. The lateral and axial resolution of conventional optical microscopy is limited by diffraction, which is typically approximately 200-300 nm. Recently, optical super-resolution techniques have been developed, such as single-molecule optical microscopy,2 saturated structured illumination microscopy,3 stimulated emission depletion microscopy,4 photoactivation localization microscopy,5,6 and stochastic optical reconstruction microscopy,7 which surpass the diffraction limit by applying concepts such as point-spread-function engineering or by utilizing the high accuracy of single-molecule localization. Thereby, a lateral resolution of 20-50 nm can be achieved and super-resolution in 3D is also feasible.
However, most of the optical techniques cannot provide information about the sample topography. Atomic force microscopy (AFM)8 allows for obtaining 3D topographical images with subnanometer resolution. Compared with other high-resolutiontechniques (e.g., electron microscopy, etc.), the particular advantage of AFM is that the measurements can be carried out in aqueous and physiological environments. Recently, the imaging speed of AFM has been dramatically improved9 from minutes to tens of milliseconds per frame, which makes it possible to film single biomolecules in action in real time.10,11 This opens the possibility to study structure, dynamics, and function of biological samples in vivo. Since structure-function relationships play a key role in bioscience, their simultaneous detection is a promising approach to yield novel insights for the characterization of biological mechanisms.
In addition to high-resolution topographical imaging, AFM can also be used for force measurements, which provide insights into the mechanical properties of cells, for example, the stiffness.12,13 Furthermore, the structural and energetic dynamics of biomolecules can be investigated by probing the interactions between a cell surface-bound molecule and a cantilever that carries a complementary binding partner, for example, another cell,14 virus,15,16 or single molecules.17-19 Ligand binding to receptors is one of the most important functional elements because it is often the initiating step in reaction pathways and signaling cascades. The high resolution of AFM in both position and force is ideally suited to gain new insights into this field. Force spectroscopy experiments probe the molecular dynamics of ligand-receptor binding, which renders it possible for estimation of affinity, rate constants, and energy barriers, as well as the bond width of the binding pocket.20-26 It also allows detection of association,27 different functional and conformational states of proteins,28 and sequential information of epigenetic modification of DNA.29