AFMs are capable of scanning biological samples that would be difficult or impossible to scan with any other type of microscope. Biomaterials, cells, and other soft samples are able to be scanned in ambient air as well as liquids to measure parameters including stiffness and adhesion.

AFM for Life Sciences Applications

Atomic Force Microscope (AFM) in Biology

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.

Imaging DNA

Imaging DNA is a key application for atomic force microscopy in life sciences because the AFM outperforms other imaging methods on multiple aspects. With our AFMs and our experience in sample preparation it is the most efficient and cost-effective method to visualize DNA constructs for research and quality control applications (from DNA in buffer to the image below in <30 minutes). In contrast to other microscopes, AFMs directly image DNA dried on a mica substrate, no stains or contrast agents required! A high purity and concentration of the DNA starting material and the reagents is important, as well as preparation expertise and a low-noise AFM setup.

 

Double-stranded plasmid DNA showing various degrees of supercoiling and a small number of DNA fragments.

Cell Mechanics

Force-distance curves consist of an approach phase where the AFM tip presses into the biological material, and a retract phase during which the tip pulls out of the material. Along the way, the applied forces bend the cantilever causing a deflection of the laser signal. This signal change and the cantilever spring constant are used to calculate the the amount of force. Read the AFMWorkshop Technical Note on Measuring Force-Distance Curves for more detailed information. AFM force measurements encompass a wide range of complexities depending on the imaging conditions (dry vs. liquid), mode (non-vibrating or vibrating), and the forces to be measured.

 

Schematic of a force-distance curve.

 

Force-distance curve taken on the periphery of a dried cell.

Correlative Imaging of Cells

An AFM mounted on an inverted light microscope delivers correlative atomic force - light microscopy data on cells. This allows users to perform established light microscopy experiments and rapidly acquire nanoscale-resolution data of subcellular features.

 

Transmitted brightfield image with inverted microscope (20x objective, bottom view).

 

In brightfield, the cell of interest has several small features on its surface (black arrowhead) that could be bacteria and a faint round structure we cannot identify (red arrow). Scanning the lower left corner of the cell with the AFM (40 x 40 um scan) surprised us: The features we thought were bacteria on the cell surface are intracellular (black arrowhead. Mitochondria?), there are several round structures of different sizes on the substrate (red arrow), and the bacteria can be identified by their height (blue arrows = cocci, blue arrowheads = bacilli).

AFM of Biomaterials

AFMs excel in the characterization of samples at the interface of materials and biology. AFM is unique in measuring quantitative three-dimensional specimen surface topography and mechanical data in a single tool and it can operate in the specific gas or liquid environment where the biomaterial fulfills its function. Statistical quantities of the specimen topography (e.g. roughness) and mechanical parameters (e.g. phase imaging and force-distance curves) can be measured and used to verify processes at the nanoscale. The AFM also differentiates itself from transmission and scanning electron microscopy (TEM/SEM) by simpler, nontoxic specimen preparation, ease of use, and substantially lower cost (you can buy one of our AFMs for the cost of an annual TEM maintenance contract).

 

Polycarbonate filtration membrane after filtering 2% milk.

 

Polymer substrate before cleaning in phase, clearly identifying a droplet contamination by a difference in stiffness.

Imaging Сells

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.

Parasites

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.

Epithelial Cells

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.

Caco-2 Cells

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