The benefits of correlative light and atomic force microscopy

Imaging cells and tissues by light microscopy (bright field, phase contrast, fluorescence, etc.) is routine in diagnostics, research and as early as elementary school. The combination of an inverted light microscope with an atomic force microscope, such as our LS-AFM, opens the door to measuring correlative features and forces with nanometer-precision and in liquid. But what are the benefits in practice? Correlative nanoscale and nanomechanical data!

AFMWorkshop LS-AFM, an atomic force microscope mounted on an inverted light microscope for correlative studies

The AFMWorkshop LS-AFM, consisting of a tip-scanning atomic force microscope mounted on an inverted light microscope (bottom view) and a video microscope (top view).

The easiest test sample: cheek cells!

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.

epithelial cheek cells in the inverted microscope of the LS-AFM, viewed with a 20x objective
epithelial cheek cells on the LS-AFM in the top-view video microscope

Left: inverted light microscope image of cheek cells taken with a 20x objective in bright field  (bottom view). Right: corresponding video microscope image (top view). Notice the outline of the probe at several millimeters distance from the surface.

epithelial cheek cells in the inverted microscope of the LS-AFM, viewed with a 20x objective
epithelial cheek cells on the LS-AFM in the top-view video microscope

The same area after approaching the AFM tip within tens of  micrometers from the surface (not in contact yet). At this distance we can aim for a specific area and initiate the final automated tip approach.

cheek cell points of interest, 20x inverted microscope
atomic force microscopy topography scan of epithelial cell from a cheek swab

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 topography scan of epithelial cell, 3d rendered and shadowed

The differences in height and shape can be illustrated by rendering and shadowing the 3d dataset

extraction of topography profiles allows precise height measurements

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).

epithelial cells on LS-AFM with contact probe in liquid

Of course the next step is to repeat this in liquid in contact mode using a contact probe, which will allow us to collect quantitative mechanical data by measuring force-distance curves.

Published data on AFMWorkshop LS-AFMs

  • Bozdag GO, Zamani-Dahaj SA, Day TC, Kahn PC, Burnetti AJ, Lac DT, Tong K, Conlin PL, Balwani AH, Dyer EL, Yunker PJ, Ratcliff WC. De novo evolution of macroscopic multicellularity. Nature. 2023 May;617(7962):747-754. doi: 10.1038/s41586-023-06052-1. Epub 2023 May 10. PMID: 37165189; PMCID: PMC10425966.

  • Deliorman M, Janahi FK, Sukumar P, Glia A, Alnemari R, Fadl S, Chen W, Qasaimeh MA. AFM-compatible microfluidic platform for affinity-based capture and nanomechanical characterization of circulating tumor cells. Microsyst Nanoeng. 2020 Mar 23;6:20. doi: 10.1038/s41378-020-0131-9. PMID: 34567635; PMCID: PMC8433216.

  • Jacobeen S, Pentz JT, Graba EC, Brandys CG, Ratcliff WC, Yunker PJ. Cellular packing, mechanical stress and the evolution of multicellularity. Nat Phys. 2018 Mar;14:286-290. doi: 10.1038/s41567-017-0002-y. PMID: 31723354; PMCID: PMC6853058.

  • Peyronnet R, Desai A, Edelmann JC, Cameron BA, 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 Nov 21;377(1864):20210326. doi: 10.1098/rstb.2021.0326. Epub 2022 Oct 3. PMID: 36189808; PMCID: PMC9527909.