Combination of High-Resolution AFM with Super-Resolution Stochastic Optical Reconstruction Microscopy (STORM)

Introduction

Since its development in 1986, atomic force microscopy (AFM) has become a versatile tool in various fields of application. As a surface imaging technique it is traditionally used in materials. Here, it is possible to resolve structures in the nanometer range with further opportunity of the investigation of material properties like friction, stifness or magnetic and electrical characteristics. Over the years the potential use of AFM in life science, e.g. biology, biophysics, biochemistry and medicine came to the force [1][2]. On the one hand, AFM as imaging method allows obtaining images in high-resolution under controled and even physiological conditions. On the other hand, AFM can be used as a force sensor to measure mechanical properties, like Young's Moduli, as well as specific interaction forces like adhesion, receptor-ligand recognition and cell-cell interactions. Especially in life science research it has to be pointed out that a combination of AFM with standard optical techniques like phase contrast microscopy and conventional fluorescence microscopy is more and more essential. A combination of both techniques opens a new world of applications, where the optics can be used as assistance as well as extension to AFM. The use of AFM together with advanced optical techniques like confocal laser scanning microscopy or TIRF microscopy is also very promising due to the better optical resolution and/or higher signal-to-background ratios of these techniques [3]. Of course, AFM and optical microscopy yield different kinds of information. While optical microscopy provides the opportunity of a specific fluorescence labeling of a structure, AFM detects the mechanical properties of the investigated samples. Down to the diffraction limit the optical and AFM image can be overlaid and correlated perfectly. Nevertheless, such a correlation leaves room for interpretation due to the different resolution ranges of both techniques. While AFM provides a nanometer resolution the conventional optical resolution is limited to a few hundred nanometers. This gap in resolution can now be filled by recently developed super-resolution techniques like Stimulated Emission Depletion Microscopy (STED), Stochastic Optical Reconstruction Microscopy (STORM) or Photo-activated Localization Microscopy (PALM), which reach an optical resolution of tens of nanometers [4].

This technical note should demonstrate the combination of AFM and STORM in a way of the general technical explanation as well as the huge benefits of a combination of both techniques.