Why use AFM to image cells and microbes?
Individual cells and microbes cover a wide range of shapes and sizes, from viruses, prokaryotes, eukaryotes, to differentiated cell types in animals and humans. In addition, cell samples can also represent highly complex environments such as biofilms, microbiomes and tissues. Embedded within are organelles, extracellular matrices and secreted molecules. The complexity is staggering and spans the nano, micro, and millimeter scales.
Our AFMs are exceptionally good at delivering rapid, inexpensive, quantitative 3D surface information of relatively flat materials (<17 µm total topography) without the need for complex sample preparation (see Comparison with SEM and TEM). AFMs can collect this information in air, other gases or liquids, from the microscale all the way to the nanoscale, and including nanomechanical measurements (see mechanobiology).
Correlative microscopy of human cells and microbiomes
Healthy human microbiomes on the other hand are critical for the homeostasis of epithelial tissues. 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. Correlative data can be obtained by AFM imaging on glass slides or coverslips after staining with histological dyes or fluorescent markers. See the LS-AFM for correlative projects.
A Gram-stained cheek swab in the inverted light microscope showing numerous bacteria. The cell in the top right corner was selected for AFM imaging, revealing a mix of cocci and bacilli on the cell surface. Note the ruffled surface of these thin, sloughed cells.
Additional data here: Specialized Applications – Correlative imaging of cells
Cells and microbiomes without correlation imaging
Epithelial cells are even easier to analyze without correlation with light microscopy. Depositing cells from a simple swab gives access to microbiome images in less than 30 minutes.
More examples of cocci and bacilli on cheek and exfoliated skin cells. Note the the skin cells lack the ruffled surface structure but show more pronounced stress fibers.
Additional data here: Specialized Applications – AFM of Microbiomes
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 or grow them on a substrate, dry, and 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. 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 synthetic materials in the environment such as the fouling of biofuel tanks and water filtration systems. 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. The intuitive design of the TT-2-AFM or the HR-AFM allow inexperienced users to scan small features such as the fimbriae below (~10 nm diameter) with just a few days of training.
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).
Additional data here: Specialized Applications – AFM of Biofilms
Mechanobiology in liquid
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 LS-AFM with other tools has been used in many amazing experiments such as cardiomyocyte mechanics, the evolution of multicellularity, and microfluidic characterization of tumor cells.
