High-resolution imaging with the Bio-AFM system NanoWizard II

Since its invention, the atomic force microscope (AFM) has been used to image a wide range of different samples at varying resolution. When the AFM was modified such that it could image samples in buffer, it became possible to address biological questions under physiological conditions with this technique. The detail in AFM images is unrivaled by other microscopy techniques that can be used to image samples in fluid, due to the signal to noise ration of the instrument. In addition, samples dried to preserve structure do not need to be further treated to generate contrast.

Atomic lattice of mica

Superior engineering and stability is required for the acquisition of high-resolution images. To demonstrate the stability of the JPK Nanowizard II even when installed on an inverted light microscope, freshly cleaved mica was imaged, in contact mode, in air. The stability of the instrument was such that one can clearly distinguish the atomic lattice of mica (Fig.1).

Fig.1: Mica imaged in contact mode. Scan size, 40 x 60 Å

Hexagonally packed intermediate layer (HPI)

The hexagonally packed intermediate (HPI) layer of the archaebacteria, Deinococcus radiodurans, has been extensively studied using atomic force microscopy [1, 2]. The HPI of D. radiodurans forms a surface layer, presumed to act as a kind of molecular sieve to regulate transport of nutrients and metabolites in and out of the cell. Data has been generated on the structure and function of the HPI layer using variety of different techniques, from biochemistry to electron microscopy. However, AFM imaging of this sample can be carried out in fluid, at high resolution. As such, dynamic changes in protein structure can be followed.

Fig.2: (A) Hpi layer patch on mica, imaged in closed loop contact mode, in fluid. (B) High-resolution image of HPI subunit pores. Circled in red is an example of a closed pore, whereas the pore circled in blue is open. Faults in the lattice are also evident, it is clear in some cases that one subunit is missing from the pore (white arrow). Image (B) kindly provided by Dr. Patrick Frederix, University of Basel.

The HPI layer is extracted from whole cells with detergent and then adsorbed to a freshly cleaved mica surface. The stable packing of the individual protein elements facilitates the acquisition of high-resolution images. The HPI layers form patches on the mica surface, and overview images of these patches already reveal the regular lattice-structure of the HPI layer (Fig 2A).

After the acquisition of an overview image of an HPI membrane patch, a suitable region can be selected for imaging at higher resolution (Fig 2B).

As the x-y positioning of the JPK Nanowizard II is controlled by a closed-loop feedback system the instrument will “zoom in” to the selected region with high accuracy. this enables the user to take fewer scans, reducing the likelihood of contaminating the tip or of destruction of the membrane patch being scanned.

Fig.3: Topographs of lambda phage DNA, imaged in ac mode in fluid. In both A and B, color scale 0 - 2 nm.

DNA imaging

Most of the data generated on the structure and function of DNA has come from the field of molecular biology. However, with the signal-to-noise ratio of AFM this fundamentally important biological molecule can be studied at high resolution in liquid and in air, to elucidate physical structure and the interaction of DNA with DNA-binding molecules.

Under appropriate conditions, DNA can be adsorbed to freshly cleaved mica and imaged in buffer. Here, we have images lambda phage DNA in ac mode in fluid (Fig. 3 and 4). The interaction of various proteins with DNA is fundamental in the processes of replication and transcription. One example is the association of DNA with histones to form nucleosomes. This condensing of DNA around the nucleosome core (consisting of a histone octamer) plays a role in the regulation of DNA replication, and transcription, as the condensed DNA is not accessible to other DNA-binding proteins.

Fig.4: AC mode topographs of DNA-nucleosomes complexes. The protein can clearly be distinguished bound along the length of the linearised pGEM plasmid. Image courtesy of Dr. Clemens Franz, Technical University of Dresden.

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