AFM Applications in Molecular Biology

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Resolving the DNA Double Helix

The BioScope Resolve uses the highly sensitive force control provided by PeakForce Tapping together with the new ScanAsyst-High Resolution probe to resolve the DNA double helix. As shown here on the left where we clearly the alternating major and minor grooves, having widths of 2.2nm and 1.2nm, respectively, in both the image and the cross section.

DNA-Double-Helix

WEBINAR: PeakForce Tapping Mode: Enabling High-Resolution Imaging of The DNA Double Helix

with guest speaker: Alice Pyne (London Centre for Nanotechnology, University College London)

High Speed and High-Resolution Imaging of Lamba Digest DNA

These four videos show consecutive series of images taken with increasing frame rates and minimal loss of resolution.  Lambda DNA was adsorbed on a mica surface and imaged in a buffer environment.

In the movie obtained at 0.5 frames per second the DNA strand is clearly resolved.   Small movements of portions of the DNA from frame to frame can be observed by following the area indicated by the red circle. Increasing the frame rate to 1, 2, and even up to 3 fps we see very minimal loss of resolution and no disruption of the DNA strands at higher imaging rates.  

DNA-origami-on-mica.png

Imaging Bacteriorhodopsin

The BioScope Resolve has also been able to obtain submolecular resolution of membrane proteins such as bacteriorhodopsin, or bR, using PeakForce Tapping – as shown in the image below. In this image we clearly see the individual molecules that make up the three subunits of the bR trimer, as shown in the inset, as well as defects within the lattice structure, as indicated by the green circle.

Lattice structure of bacteriorhodopsin

It is important to remember that both of the images shown above were obtained while operating the BioScope Resolve AFM on an inverted light microscope – and not as a stand-alone configuration.

The FastScan BioAFM provides sub-molecular resolution biological AFM imaging while scanning at speeds 100 times faster than a typical BioAFM.  The atomic resolution capabilities of FastScan BioAFM are demonstrated in this image of Bacteriorhodopsin trimer structure.  The image on the right shows raw data from one single image, not the result of multiple image averaging which is typically performed with other high-resolution imaging AFMs. 

Molecular imaging of bacteriorhodopsin in buffer

High-Resolution Imaging of Origami DNA at High Speed

Pixel resolution: The top 3 images show triangular-shaped DNA origami imaged in buffer solution using a typical pixel resolution of 512 pixels.  Other high-speed AFMs often reduce the number of pixels in an image to 100 or less to achieve higher scan rates, thereby requiring very small scan sizes to minimize the loss of resolution due to larger pixel sizes.   There is a lower limit to the number of pixels used to acquire an image before the features of interest in your image can no longer be viewed.


Imaging speed: Traditional AFM images of DNA origami obtained at this high of a pixel density are typically conducted at 1 or 2 Hz, which results in each image taking several minutes to capture.  FastScan-Bio clearly shows the individual DNA strands that make up each piece of origami, as well as the single stranded DNA end piece observed on the side of at least 2 of the triangles, at scan rates of 11Hz to 43Hz without loss of resolution.

 

 

DNA-highspeed-highresolution.PNG
Origami DNA in buffer: (350nm x 350nm x 3.4nm). Acquisition times at the given frequencies were: 11Hz = 46sec, 22Hz = 23sec, 43Hz = 12sec, 49Hz = 5sec, 87Hz = 3sec, 98Hz = 2.5sec. Sample courtesy of P. Rothemond & L. Qian, CalTech, USA.


In order to image the DNA imaging process even faster, the pixels count was decreased from 512 lines per image to 256 lines per image.  This parameter change allowed us to further increase the scan rate up to 98Hz, which is nearly 100 times faster than traditional AFM DNA origami data acquisition.  The end result is that imaging that formerly took minutes can be performed in a matter of seconds.  The images taken at these high speeds show very well resolved origami triangles, including the single stranded piece of DNA on the sides of the triangles.