Force-feedback high-speed atomic force microscope for studying large biological systems

Abstract

We designed and developed a high-speed atomic force microscope (HSAFM) utilizing a force-feedback scheme for imaging large biological samples. The system collects three simultaneous images: a deflection image, a topographic image, and a force image. We demonstrated that this force-feedback HSAFM is capable of acquiring large topographic images of Escherichia coli biofilms at approximately one frame per second in air. We discuss how the self-actuating cantilever and the piezo tube follow those larger biological topographic features during the HSAFM imaging process.

Introduction

The recent advancement of high-speed atomic force microscopy (HSAFM) has enabled researchers to view the nanometer-scale dynamic behavior of individual biological and bio-relevant molecules at a molecular-level resolution under physiologically relevant time scales (Ando et al., 2001, Crampton et al., 2007, van Noort et al., 1999), which is the realization of a dream in the life sciences. Such studies include the visualization of various dynamic activities carried out by biological macromolecules (Ando et al., 2008a, Ando et al., 2008b), such as motor proteins and cytoskeletal fibers (Ando et al., 2001), nucleic acids/proteins in real time (Crampton et al., 2007), and biopolymers (van Noort et al., 1999, Viani et al., 1999). The enhancement of the scan speed, development of a high z-bandwidth feedback loop, and the advancement of tapping-mode imaging with a small cantilever make it possible to improve the resolution of topographic signals in both time and space in less invasive ways (Ando et al., 2001, Ando et al., 2008a, Ando et al., 2008b, Crampton et al., 2007, Humphris et al., 2005, Kodera et al., 2006, Picco et al., 2007, van Noort et al., 1999, Zou et al., 2008).

These high-speed imaging applications now extend to the cellular/bacterial systems with the use of a smaller cantilever (Fantner et al., 2010, Hansma et al., 2006). Fantner et al. viewed that the limitation originates from the size of the cantilever, as the cantilever response speed is proportional to the cantilever resonance frequencies (Viani et al., 1999, Fantner et al., 2010). The use of a cantilever with high-resonant vibrational frequency allows more rapid vertical tip movement, thus obtaining high-speed imaging. By reducing the size of the cantilevers of HSAFM to a width of 10 μm and a length of 20–30 μm (Fantner et al., 2010), instead of using conventional cantilevers that are tens of μm wide and hundreds of μm long, the system has demonstrated image speeds up to one frame per second for small biomolecular structures such as DNA and one frame per ten seconds for larger biological systems, such as bacteria (Fantner et al., 2010, Hansma et al., 2006, Viani et al., 1999). To understand many rapid large-scale biological phenomena, this imaging speed is not sufficient enough. For instance, the roughening variation of bacteria occurs within a few seconds in response to an antimicrobial (Fantner et al., 2010, Katan and Dekker, 2011). Also, the use of smaller cantilevers creates some challenges, such as fabrication and signal detection with a smaller laser spot size (Ando et al., 2001, Walters et al., 1996).

Here we introduce an alternative approach to that of employing a small cantilever for high-speed imaging, called “force-feedback” HSAFM. This new system utilizes our recent development of a cantilever-based optical interfacial microscope (COIFM) (Bonander and Kim, 2008, Kim et al., 2011a, Kim et al., 2011b). Rather than using a smaller cantilever, we still use a conventional-size self-actuation cantilever that is capable of fast response through the force-feedback mechanism. The force-feedback shortens the response time of the sensor, which is the most essential component for this HSAFM. Not only does it allow for the high-speed imaging, it has a displacement capability of micrometers through a piezo tube, thus enabling the system to extend the applications ranging from biomolecules to cellular systems. This novel force-feedback HSAFM will contribute greatly to the studies of these large-scale biological phenomena (Fantner et al., 2010, Hansma et al., 2006, Katan and Dekker, 2011) through the improvement of the time resolution for scan sizes up to several micrometers.