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BioScience AFM – Capturing Dynamics from Single Molecules to Living Cells


Dimitar R. Stamov , 1 Stefan B. Kaemmer , 2 * Anne Hermsdörfer , 1 Jörg Barner , 1 Torsten Jähnke , 1


and Heiko Haschke 1 1 JPK Instruments AG , Colditzstr. 34-36 , 12099 Berlin , Germany 2 JPK Instruments USA , 4189 Carpinteria Ave. , Suite 1, Carpinteria , CA 93013


* stefan.kaemmer@jpk.com


Introduction T e last three decades have seen the rise of the atomic force microscope (AFM) as an indispensable tool for high- resolution structural analysis of specimens ranging from single molecules [ 1 ] to complex biological systems such as proteins and cells [ 2 ]. Unlike other high-resolution imaging techniques, such as advanced electron microscopy and super-resolution light microscopy, AFM remains the only tool that currently off ers premium resolution of the analyzed biological systems while being able to simultaneously acquire information about the sample’s mechanical properties at near physiological/native sample conditions. An additional benefi t of AFM is that by default it also does not demand any sample modifi cation and therefore does not introduce preparation artifacts. T e two most commonly used AFM modes for investigat- ing biological samples in a near-native environment employ either dynamic or static AFM tip motion (see Figure 1 ). T e dynamic modes, in which a vibrating cantilever is driven over a surface with a defi ned amplitude, are preferred for soſt bio- samples because of the less invasive nature of the imaging. Even though repulsive forces are commonly used, resonance dyna mic modes generally apply lower forces to the specimen during the imag- ing process. Pure non-contact techniques are less frequently used with biological samples because of the rather complex composition of the imaging buff ers and media used.


Studying single macromol- ecule dynamics and the function of complex biological systems, such as individual living cells, requires a tool that can provide both high spatial and high temporal resolution [ 3 ]. Developments in the last 10–15 years have paved the way toward the application of ultra-small cantilevers, piezoac- tuator-based sample scanners, and optical beam defl ection (OBD) detectors for studying high-speed single-molecule processes [ 4 – 6 ]. Such high-speed developments


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are rarely applicable for living cells as they exhibit a signifi - cantly reduced xy -scan window of only a few microns and a z -range of less than a micrometer [ 7 ]. Recent developments in fast tip-scanning AFMs [ 8 ] allow the successful structural analysis of a number of dynamic processes in cells such as exocytosis, vesicle transport, cytoskeleton reorganization, and cell migration, all taking place on the timescale of seconds. Structurally resolving morphological surface changes and the above-mentioned cellular events are no longer limited by the fundamental diff raction limit inherent to conventional light microscopy. It is the combination of light microscopy and AFM that leverages the advantages of optical/fl uorescence detection systems enabling truly correlative AFM [ 9 ]. T is article describes some examples of these techniques.


Materials and Methods


Combining light optical microscopy and AFM . Combining AFM with optical information has become a standard when working with biological samples above Abbe’s resolution limit, as well as with single molecules carrying immunolabels. T e convenience of combining high-resolution AFM information from an optically


Figure 1 : Conventional AFM imaging modes. (a) The most commonly used dynamic AFM mode is AC in which a cantilever is oscillated with a certain amplitude close to its resonance frequency and intermittently touches the surface. (b) Static AFM imaging (contact mode) maintains a constant defl ection of the cantilever (pre-set force) to the sample while raster scanning and without detaching from the surface. (c) Quantitative imaging allows recording of a complete force curve (vertical application of a pre-set force in approach/retract regime) at each pixel. (d) Further monitoring of amplitude damping or phase shift during AC imaging enables related amplitude and phase modulation modes. (e) Simultaneous acquisition of the cantilever-sample interaction during the force spectroscopy-based QI mode enables height determination, adhesion, or mechanical characterization.


doi: 10.1017/S1551929515001005 www.microscopy-today.com • 2015 November


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