Looking at living cells down to individual molecules

August 5, 2015

3D rendered correlative AFM/PALM image of a fixed mammalian cell (mouse embryonic fibroblast (MEF) cell) expressing the fusion protein paxillin-mEOS2 (credit: Pascal D. Odermatt et al./Nano Letters)

EPFL scientists have captured images of living cells with unprecedented nanoscale resolution — even the evolution of their structure and molecular characteristics.

They did that by combining two cutting edge microscopy techniques — high-speed atomic force microscopy and a single-molecule-localization, super-resolution optical imaging system — into one instrument.

Their work was published in the journal ACS Nano Letters.

The “correlated single molecule localization microscope” combines two methods:

  • An atomic force microscope (AFM) “feels” the surface being observed using a tiny force sensitive needle, capturing the 3D structure. It is installed above the sample.
  • Meanwhile, the microscope technique, known at PALM (photo-activated localization microscopy), observes the sample from below. It selectively stains the sample with fluorescent molecules to label certain selected molecules by making them blink, and then follows their path in the interior of a cell. Its inventors were awarded the Nobel Prize last year.

The scientists also developed special software that assembles the images from the two instruments, providing a precise 3D visualization of the observed sample.

Correlative AFM-SMLM: instrument setup. (a) Schematic of the aligned optical path with the AFM cantilever. By laterally translating the incoming laser beam using a micrometer screw, the TIRF illumination condition is enabled. The AFM cantilever is centered in the field of view by adjusting the position of the inverted optical microscope mounted on an x/y-translation stage (as shown in b and c). (b) Mechanical integration of an inverted optical microscope and the AFM. The inverted optical microscope is mounted on an x/y-translation stage. Around it a mechanical support structure is built to hold the AFM in place without mechanically contacting the microscope body. The whole instrument is placed on a vibration isolation platform inside an acoustic isolation box. (c) Photograph of the instrument and (d) zoom in to the AFM cantilever aligned to the optical axis. (credit: Pascal D. Odermatt et al./Nano Letters)

By taking successive images of the same living cell, the scientists were, for the first time ever, able to follow the behavior of protein clusters in relation to the 3D structure of the cell. “That could, for example, allow us to observe the inner workings of cell division, or unravel how stem cells react to mechanical forces” says Henrik Deschout, post doctoral researcher in EPFL’s Laboratory of Nanometer-Scale Biology, which is directed by Aleksandra Radenovic.

The prototype stage has already attracted the interest of many other researchers as well as leading microscope manufacturers. The microscope could be of great interest to researchers in cellular-, micro- and mechanobiology, allowing scientists to shed new light on the intricate mechanisms occurring in living cells, the researchers say.


Abstract of High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy

Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.