Applications

Imaging cells with AFM

You are probably on this webpage because you are trying to figure out what information you can get from measuring cells with an atomic force microscope and what these systems really deliver. So let’s jump right in!

Which cells, which features, what is AFM good for?

Cells come I various shapes and sizes, prokaryotic or eukaryotic, specific cell type, developmental stage, and larger context.1 Within that are organelles and molecules, and the research question, which leads to the question of methods. The trick to AFM is designing your experimental approach in the most efficient way to answer your question.
AFMs are exceptionally good at delivering rapid, inexpensive surface information of relatively flat materials (<15 µm total topography) and can collect this information in air, other gases or liquids, from the microscale all the way to the nanoscale, and including all kinds of nanomechanical measurements. (Comparison to SEM and TEM below.)
The key differentiators between our AFMs is that the HR-AFM excels at nanoscale measurements, whereas the LS-AFM is ideal for correlative imaging with AFM and light microscopy.

Isolated microbes

Working with isolated or cultured microbes is the easiest starting point both in terms of biological laboratory practice and AFM imaging. Microbes are widely used in biosynthesis applications in research and biomanufacturing and easily produced in large numbers. Standard liquid cultures and colonies on agar plates can easily be spread on a substrate, air dried and imaged directly to assess uniformity of the culture and identify potential contamination. These images are easy to collect in about 30 minutes! Transfer the cells to a substrate, let dry, image.

Single bacterial colony with uniform cells picked directly from an agar plate, dried and imaged without further sample preparation (topography, 50x50x0.9 µm volume).

Biofilms

Microbial communities play key roles in medical and environmental contexts and create complex structures in the form of biofilms and entire microbiomes.2,3 Biofilms are now recognized as a key feature of the bacterial life cycle with wide-ranging implications in infections and their treatment in humans, animals and plants, but also bacterial attachment to medical devices and man-made materials in the environment such as the fouling of biofuel tanks and water filtration systems.4 Even with simple air-drying and a few AFM scans the samples below reveal the intricate micro- and nanostructures that give the biofilm its resilience to chemical and mechanical disruptions. Even scanning small features such as the fimbriae below (<10 nm diameter) is straightforward using the HR-AFM.

This sample is from a bacterial biofilm found in a CPAP machine. The biofilm was mechanically disrupted and illustrates the extensive matrix (pockets) that surrounds the bacteria (topography, 20x20x1.2 µm volume).

High resolution phase image of the same bacteria embedded in the extracellular matrix (5×5 µm scan size).

fimbriae in a bacterial biofilm rendered, shadowed and height color coded

Shaded surface rendering of  fimbriae network within the biofilm.

Human cells and microbiomes

Healthy human microbiomes on the other hand are critical for the homeostasis of epithelial tissues.5 When studied using genomic approaches these microbiomes are highly complex, which is why Gram staining for light microscopy is commonly used for research and diagnostic purposes. Here again AFM is very easy to use thanks to the very flat nature of sloughed epithelial cells with the attached microbiome. Even more interesting is that AFM imaging can be performed on glass slides after gram staining (before coverslipping) to obtain correlative data. See the LS-AFM for correlative projects.

Oral microbiomes, cocci and bacilli on epithelial cells from cheek swabs.

Skin microbiomes, cocci and bacilli on exfoliated skin cells.

More Images of Cells and Cellular Structures

Advanced methods

The examples above illustrate how AFM excels in simplicity and cost for imaging cells after fixation and/or drying. But the two differentiating capabilities of AFM are the option to scan live cells in liquid and to measure a range of nanomechanical parameters, such as stiffness, adhesion forces, unfolding, and many more. It must be noted that while possible, these measurements become more difficult to execute in liquid and often are much harder to interpret. Nevertheless, this key advantage and the ability to integrate the AFM with other tools has been used in many amazing experiments. 6–8

Measurements in liquid can be performed using the dunk and scan option on the HR-AFM or with the LS-AFM, where the AFM is mounted on the stage of an inverted microscope for correlative microscopy.

E. coli cell scanned in liquid
Aiming for cells in brightfield on the LS-AFM
Neutrophil on the LS-AFM

AFM vs. SEM and TEM

The scanning electron microscope (SEM) is a good choice to analyze biomaterials but typically requires metal coating of the fully dried, nonconductive biological sample to achieve high resolution with sufficient contrast. Once prepared and loaded into the vacuum chamber, the SEM can quickly collect many images in a short period of time and collect elemental distribution data with an EDS system. The SEM excels in imaging small features on samples with large underlying topography thanks to its long focal depth and does this seamlessly from 50x to 500,000x magnification. These capabilities come at substantial cost, requiring access to equipment with >$300k purchase price and >$20k in annual maintenance fees that is only accessible to users of high-end facilities.

The transmission electron microscope (TEM) is an excellent option for imaging thin (<1 µm) biomaterials such as macromolecules and sectioned cells and can image internal structures. The downside is that contrast formation either relies on the use of toxic heavy metal stains or alternatively very complex cryo-electron microscopes. This comes at even higher cost than SEM, >$700k purchase price and >$30k in annual maintenance.

The AFM does not require any sample processing because the probe tip directly interacts with the sample surface. A sample is immobilized on a substrate (mica or glass slides), mounted onto an AFM disc with double-sided tape and the first image is collected within minutes. For liquid and correlative live imaging, standard glass slides can be used. AFM delivers quantitative 3D data, can image surfaces in liquid and measure or exert forces through the direct interaction with the specimen. Compared to SEM/TEM, our AFM systems used here deliver this data at a >10x lower purchasing cost without the need for costly maintenance contracts, and without the use of toxic heavy metal stains. The scans are not instantaneous but take a few minutes to record depending on the selected parameters and AFM cannot scan surfaces with large topography >15 µm.

References

  • 1

    Cell (Biology). Wikipedia; 2023.

  • 2

    Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2 (2), 95–108. https://doi.org/10.1038/nrmicro821

  • 3
    Microbiome. Genome.gov. https://www.genome.gov/genetics-glossary/Microbiome (accessed 2023-12-04).
  • 4
    Muhammad, M. H.; Idris, A. L.; Fan, X.; Guo, Y.; Yu, Y.; Jin, X.; Qiu, J.; Guan, X.; Huang, T. Beyond Risk: Bacterial Biofilms and Their Regulating Approaches. Front. Microbiol. 2020, 11.
  • 5
    Reynoso-García, J.; Miranda-Santiago, A. E.; Meléndez-Vázquez, N. M.; Acosta-Pagán, K.; Sánchez-Rosado, M.; Díaz-Rivera, J.; Rosado-Quiñones, A. M.; Acevedo-Márquez, L.; Cruz-Roldán, L.; Tosado-Rodríguez, E. L.; Figueroa-Gispert, M. D. M.; Godoy-Vitorino, F. A Complete Guide to Human Microbiomes: Body Niches, Transmission, Development, Dysbiosis, and Restoration. Front. Syst. Biol. 2022, 2.
  • 6
    Peyronnet, R.; Desai, A.; Edelmann, J.-C.; Cameron, B. A.; Emig, R.; Kohl, P.; Dean, D. Simultaneous Assessment of Radial and Axial Myocyte Mechanics by Combining Atomic Force Microscopy and Carbon Fibre Techniques. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2022, 377 (1864), 20210326. https://doi.org/10.1098/rstb.2021.0326.
  • 7
    Bozdag, G. O.; Zamani-Dahaj, S. A.; Day, T. C.; Kahn, P. C.; Burnetti, A. J.; Lac, D. T.; Tong, K.; Conlin, P. L.; Balwani, A. H.; Dyer, E. L.; Yunker, P. J.; Ratcliff, W. C. De Novo Evolution of Macroscopic Multicellularity. Nature 2023, 617 (7962), 747–754. https://doi.org/10.1038/s41586-023-06052-1.
  • 8
    Deliorman, M.; Glia, A.; Qasaimeh, M. A. Affinity-Based Microfluidics Combined with Atomic Force Microscopy for Isolation and Nanomechanical Characterization of Circulating Tumor Cells. Methods Mol. Biol. Clifton NJ 2023, 2679, 41–66. https://doi.org/10.1007/978-1-0716-3271-0_4.