Correlation with brightfield LM
A simple cheek swab can be used to deposit cheek epithelial cells (stratified squamous epithelium) on a microscope slide or coverslip. These swabs make for very interesting imaging as they also include the person’s oral microbiome and a range of compounds present in saliva. The cheek cells represent keratinized, mostly dead cells that have lost most of their nucleus and organelles, making them very flat and ideal for AFM.
The open architecture of the LS-AFM allows the user to independently modify and collect data from their inverted microscope setup (bright field, fluorescence, spectroscopy, …), and use the top-view video microscope to position the AFM probe onto the same feature.
Transmitted brightfield image with inverted microscope (20x objective, bottom view).
Reflected brightfield image with video microscope (zoom lens, top 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).
Because the AFM collects quantitative 3D data, we can also extract line profiles and make accurate measurements of the features we observe. The bacteria are ~500 nm tall, and the mystery round features appear to be aggregates of smaller particles in the range of tens of nanometers in height (caveat, this is an air-dried specimen, it is likely that some of the features have collapsed during drying).
Combination with staining protocols
Cheek swabs are compelling samples because they come with their own microbiome. The number and types of bacteria vary between people and dramatically increase after easting. To better visualize the many bacteria we performed a Gram-stain on the dried cells. The same concept can be used with other stains or fluorescent markers (e.g. antibody staining).
Gram-stained cells in the inverted microscope showing numerous bacteria on the glass slide (20x objective, bottom view).
AFM image of the single cell in the top right quadrant. Both cocci and bacilli are seen on the cell surface. The ruffled surface (~50 nm height) is clearly visible at the cell periphery (cell thickness ~500 nm).
Topography image with line profiles.
Extracted profiles showing the height of membrane ruffles and dividing cocci.
3D visualization highlighting all surface features.
Live cell correlation
Correlative AFM-LM can be extended to studying live cells, correlating events captured by light microscopy to nanoscale topography information acquired with the AFM. In liquid the AFM also provides the unique capability to perform mechanobiology assays.
Brightfield image of a Caco-2 cell in buffer, aiming the probe to scan the boxed region.
Corresponding topography of the boxed region by AFM.
Mechanobiology of cells in liquid.
Correlative light and atomic force microscopy setup
Imaging cells and tissues by light microscopy is routine in diagnostics, research and as early as elementary school. The combination of an inverted light microscope with a tip-scanning atomic force microscope, such as the LS-AFM, allows users to make nanometer-precision measurements on their standard samples mounted on glass slides or coverslips.
