QUESANT Applications Note No. AN-3

Biological Applications and Liquid Scanning

       Atomic Force Microscopy adds an entirely new dimension to microscopy. AFM is able to give you not just 2-D images of a surface like an SEM, but instead can provide 3-D topographic images that yield a measure of surface roughness on the nanometer scale. AFM is unique in its ability to image biological samples under very high resolution conditions (as is required for some proteins and DNA) because it can acquire the data while the sample is in a biologically relevant medium.

- sample as seen through as scanning probe microscope (SPM) and atomic force microscope (AFM)

       The Quesant AFM can scan samples in a liquid medium with relative ease. This powerful capability opens up a whole new world for microscopists in the field of biology, because
entire cells can be scanned while they are still alive and active in their particular function. In addition to scanning an image, the AFM is a tactile tool that can physically probe a specimen by means of a force-distance curve (F-D). The F-D curve is essentially a low-force loading experiment that can also provide valuable information on sample binding properties.

- sample as seen through as scanning probe microscope (SPM) and atomic force microscope (AFM)

Figures 1 & 2 show AFM images of the cell-adhesion protein, fibronectin, as deposited onto a glass cover slide. These images were obtained in contact mode while in PBS. Fibronectin promotes binding of cellular materials within the extracellular matrix. As such, AFM studies that use both imaging and F-D curves, can help determine the binding forces between fibronectin and extracellular materials, and between synthetic biomaterials. Figure 1 shows a continuous layer of fibronectin that was obtained on the first scan frame. After scanning a few frames, the protein layer is disturbed, even though the cantilever tracking forces are less than 25 nanoNewtons. Controlling the tracking force of the cantilever while scanning in a liquid medium is generally straight forward, because the meniscus forces that are usually present between the probe tip and sample under ambient conditions are no longer present in liquid. Consequently, a liquid medium is also a more useful environment for F-D curves.

Plasmid DNA on mica - sample as seen through as scanning probe microscope (SPM) and atomic force microscope (AFM)

       The Cancer Research Institute at Rutgers University is currently developing a method to quantify fluorescent signals from the in-situ hybridization of DNA (FISH). The sample from Figure 3 represents the control sample for the FISH experiment, and this AFM image verifies the presence of single-plasmid DNA. Signals obtained by FISH were interpreted to represent mostly single plasmids (" 70% or so), but with the presence of a significant number of groups of two, and even three plasmids. With the high resolution power of AFM, the plasmid DNA is directly resolved and thus can be used to verify the conclusions derived from the FISH experiments. In order to verify the FISH experiments, it is necessary to image, on a random basis, about 100 plasmids by AFM.

Human hair - samples as seen through as scanning probe microscope (SPM) and atomic force microscope (AFM)

       Figure 4 is an example of using AFM to study the effect of consumer products on biological materials. In this case, a hair fiber has been loaded with hair-care products, which remain as residual deposits concentrated in the cuticle structure. LFM data show that the residue has a lower frictional response than the hair fiber, because that area has a darker image contrast.