Gentle High Speed Imaging of Live Cells with AFM

The imaging rate of conventional atomic force microscopy (AFM) is too low to capture the dynamic behavior of biomolecules. To overcome this problem, we have been developing various devices and techniques, including small cantilevers and high-speed scanners. The feedback bandwidth in the tapping-mode now exceeds 100 kHz and hence the maximum possible imaging rate reaches 25 frames per sec (fps). Importantly the tip-force exerting onto the sample is dramatically reduced. Thus, it is now possible to take video images of dynamically moving protein molecules in action without disturbing their function, including walking myosin V molecules along actin tracks.

Micron, 2012

In this study, we demonstrate the increased performance in speed and sensitivity achieved by the use of small AFM cantilevers on a standard AFM system. For this, small rectangular silicon oxynitride cantilevers were utilized to arrive at faster atomic force microscopy (AFM) imaging times and more sensitive molecular recognition force spectroscopy (MRFS) experiments. The cantilevers we used had lengths between 13 and 46 m, a width of about 11 m, and a thickness between 150 and 600 nm. They were coated with chromium and gold on the backside for a better laser reflection. We characterized these small cantilevers through their frequency spectrum and with electron microscopy. Due to their small size and high resonance frequency we were able to increase the imaging speed by a factor of 10 without any loss in resolution for images from several m scansize down to the nanometer scale. This was shown on bacterial surface layers (s-layer) with tapping mode under aqueous, near physiological conditions and on nuclear membranes in contact mode in ambient environment. In addition, we showed that single molecular forces can be measured with an up to 5 times higher force sensitivity in comparison to conventional cantilevers with similar spring constants.

MRS Proceedings, 2007

High speed atomic force microscopy (AFM) holds the promise of investigating dynamic systems in real time with single molecule resolution. With the big push towards understanding more complex systems such as cell mechanics or cell-cell and cell-virus interactions, a tool is required that can extract information about these processes in real time in a physiological environment. Atomic force microscopy has been successfully used for investigations of many biological systems and materials in real life conditions, but taking AFM images takes too long to follow many biologically relevant processes. Therefore, attempts have been made to develop high speed AFM by reengineering all the components of an AFM system and much progress has been made. To be useful for investigations of biological systems however, it is often essential to keep imaging forces low in order to get good image quality and not to damage the sample. In this paper we will discuss new small AFM cantilevers we've develop...

Microorganisms, 2021

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Pflügers Archiv - European Journal of Physiology, 2007

The exploration of molecular processes governing physiological functions has significantly benefited from the emergence of novel nanoscaled techniques. Atomic force microscopy in force measurement mode can be used to investigate a multitude of cellular events in individual living cells with great sensitivity. Precise regions of the plasma membrane can be examined in relation to particular signalling pathways activated by a mechanical stimulus. Similarly, subtle cellular movements induced by biochemical activation of specific membrane receptors can be detected in real time with excellent temporal and spatial resolution. The possibility to challenge locally and mechanically cell surface receptors also provides information on the control exerted by a cell over individual adhesion sites. Overall, this information is vital for an in-depth understanding of mechanisms related to cellular movement and morphological regulation. Lastly, atomic force microscope-based nanomanipulations on living cells have recently been proposed as a tool to influence and monitor cellular homeostasis by introducing specific molecular entities into or extracting them from the cytoplasm of individual cells. This review provides detailed examples on how such atomic force microscopy experiments can be conducted to investigate processes relevant to cell physiology.

Nature Methods, 2013

Biological systems, ranging from single molecules to cells and tissues, have heterogeneous structural, biophysical and chemical properties that change dynamically to fulfill versatile functions. At the nanoscale, these properties determine how the highly sophisticated molecular machinery of the living cell works and responds to its complex environment. Hence, there is an urgent need for methods that can image native biological systems in physiological buffer and simultaneously map the systems' multiple properties at molecular resolution.

Optics Letters, 2007

We report a novel optical-tweezers-based method to study the membrane motion at the leading edge of biological cells with nanometer spatial and microsecond temporal resolution. A diffraction-limited laser spot was positioned at the leading edge of a cell, and the forward scattered light was imaged on a quadrant photodiode that served as a position sensitive device. The universality of this technique is demonstrated with different cell types. We investigated the membrane motion at the leading edge of red blood cells in detail and showed that this technique can achieve simultaneous manipulation and detection of cellular edge dynamics with unprecedented precision.

Nature Protocols, 2014

protocol nature protocols | VOL.9 NO.5 | 2014 | 1113

Annual review of biophysics, 2013

Directly observing individual protein molecules in action at high spatiotemporal resolution has long been a holy grail for biological science. This is because we long have had to infer how proteins function from the static snapshots of their structures and dynamic behavior of optical makers attached to the molecules. This limitation has recently been removed to a large extent by the materialization of high-speed atomic force microscopy (HS-AFM). HS-AFM allows us to directly visualize the structure dynamics and dynamic processes of biological molecules in physiological solutions, at subsecond to sub-100-ms temporal resolution, without disturbing their function. In fact, dynamically acting molecules such as myosin V walking on an actin filament and bacteriorhodopsin in response to light are successfully visualized. In this review, we first describe theoretical considerations for the highest possible imaging rate of this new microscope, and then highlight recent imaging studies. Finall...

Journal of Molecular Recognition, 2011

Atomic force microscopy (AFM) investigations of living cells provide new information in both biology and medicine. However, slow cell dynamics and the need for statistically significant sample sizes mean that data collection can be an extremely lengthy process. We address this problem by parallelizing AFM experiments using a two-dimensional cantilever array, instead of a single cantilever. We have developed an instrument able to operate a two-dimensional cantilever array, to perform topographical and mechanical investigations in both air and liquid. Deflection readout for all cantilevers of the probe array is performed in parallel and online by interferometry. Probe arrays were microfabricated in silicon nitride. Proof-of-concept has been demonstrated by analyzing the topography of hard surfaces and fixed cells in parallel, and by performing parallel force spectroscopy on living cells. These results open new research opportunities in cell biology by measuring the adhesion and elastic properties of a large number of cells. Both properties are essential parameters for research in metastatic cancer development.