AFM for Life Sciences Applications
Atomic force microscopes are capable of making measurements on biological samples at the nanoscale that are difficult or even impossible with any other type of microscope. AFM allows the nanoscale imaging of soft biomaterials including cells and DNA in both ambient atmospheric conditions as well as liquid environments, Examples of biology applications which are unique to atomic force microscopes are shown below.
Such high resolution imaging requires an instrument noise floor of less than 0.1nm, a good tip approach leading to the maintenance of a sharp tip, the capability to scan with very light forces, and placement of the microscope in an environment with minimal structural and acoustic vibrations.
Proper sample preparation is another critical factor. Sample preparation techniques are described in Atomic Force Microscopy by Eaton and West.
Atomic force microscopy has a particular advantage over electron microscopy, in that cells and biomaterials can be imaged in partially or totally hydrated conditions including ambient air and liquid environments.
25 µm x 25µm image of parasites measured in air.
These Leishmania cells have been treated with an antimicrobial peptide, leading to highly roughened cell membranes, which can be measured and quantified by AFM.
32 µm x 32 µm image of epithelial cells measured in liquid with an AFMWorkshop Dunk and Scan.
Measurement of high resolution images of cells in liquid (e.g., under physiological conditions) is another possibility unique to AFM, and can give much more relevant results than electron microscopy, which requires cell fixation, leading to artifacts.
Bacteria Spore Mutants
30 µmx 30 µm image of bacteria spore mutants.
The ability to image a very large number of cells, such as these spores, allows the researcher to obtain statistically relevant information about a population of cells. Images of multiple cells can be also useful to assess inter-cellular effects, such as clustering and adhesion.
Imaging cells in combination with an inverted optical microscope
The inverted optical microscope facilitates direct placement of the probe on an area of interest for scanning. Additionally the inverted microscope can be operated in epifluorescence mode.
Neutrophil A Cells
Inverted optical microscope image of neutrophil A cells. The dotted outline is the area scanned with the AFM.
Light Shaded AFM image of the cells visualized in the optical microscope image.
Inverted optical microscope image of Caco-2 cells in the LS-AFM. Clearly visible is the AFM cantilever on the right side of the image. A box identifies the area for AFM scanning.
3-D color scale image of the Caco-2 cell. The scan range is 48 µm x 48 µm.
CACO-2 cell structure in the presence of low concentration of quantum dots. Left: Epifluorescence, showing brightfield (red), DAPI (blue), 2.2nm quantum dot PL emission at 560 nm (green). Right: Topographic AFM image of the indicated area.
Measuring Stiffness of Biomaterials at the Nanoscale
Monitoring the deflection of a cantilever as it is pushed against a sample results in a force/distance curve. From the force distance curve many parameters may be measured, such as stiffness of the sample and probe-sample adhesion.
In biological samples, the most common application is measurement of intermolecular forces. For example, this could be used to measure the interaction force between an antigen and an antibody directly. Cell-cell adhesion forces and cellular stiffness can also be measured.
The above screen shot demonstrates Advanced Force Distance Curve software measuring an AFM image.
1. Force-Distance data display region
2. Slider indicates the extension of the Z piezoelectric ceramic
3. Control parameter selection options
4. AFM Image for selecting locations for force-distance measurements
Recommended AFM products for life sciences applications:
TT-2 Atomic Force Microscope ‒ Measurements of biomolecules and biomaterials
LS-Atomic Force Microscope ‒ Imaging cells and measuring stiffness/adhesion