Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology
Preprint posted on 21 October 2019 https://www.biorxiv.org/content/10.1101/812396v2
The advent of Atomic Force Microscopy (AFM) in biology revolutionized the way we understand cells as mechanical-responsive elements. Classically, at the cellular level, the AFM technique is used to probe viscoelastic properties and measure adhesion forces. In the most common AFM setups the AFM head is mounted in an epifluorescence microscope allowing the user to monitor the sample. However, due to the AFM design which applies the forces perpendicular to the image plane, the imaging data capture has poor z-resolution making it difficult to catch subtleties as cellular deformations. In the meanwhile, combining force spectroscopy with fluorescence high-resolution imaging in 3D has proven to be challenging due to the complex optics setup and the extremely low vibrations that the AFM measurements tolerate. This preprint reports a novel approach to tackle this problem: an integrated system that combines a vertical Line Bessel light sheet system (LSFM) with an AFM module.
By using a Line Bessel version of the previously described (Beicker et al., 2018) microscope equipped with a PRISM+ (pathway rotated imaging for sideways microscopy) the authors describe a multimodal approach that allows two-colour, 3D live cell-imaging and consecutive AFM spectroscopy data synchronization. The new LSFM-AFM microscope is a single-objective non-diffractive version which includes a fully-computer controlled light path, composed of several electronic tuneable lenses (ETL) and steering mirrors. This approach allows for high-frame rate image acquisition and remote focusing capabilities while minimizing the working vibration (Figure1)(Nelsen et al., 2019).
+ PRISM principle: A 45°reflecting optical component is brought next to the cell of interest. Upon the approaching of the objective towards the sample the focal plane is subsequent move in the z-direction until intersects the 45°reflecting optical component. There the virtual x-z image is formed.
Figure1: (a) Schematic representation of LSFM-AFM microscope and (b) PRISM with x-z image projection (c) Side-view of fluorescent labelled RAW 264.7 macrophage (green) and cantilever tip coated with IgG (magenta). Adapted from Nelsen et al., 2019. Courtesy of the authors.
Proof of principle
In order to test the different capabilities of the LSFM-AFM microscope, the authors performed successfully several proof of principle experiments such as:
- Imaging lysosome trafficking under controlled mechanical load (Live cell two-coloured volumetric imaging)
- Force measurements of macrophage phagocytosis (Live cell two-coloured imaging with AFM force data synchronization)
- Force measurements of macrophage phagocytosis in 3D (Live cell two-coloured volumetric imaging with AFM force data synchronization) (Figure2)
Figure2: (a) F-actin dynamics during RAW 264.7 macrophage phagocytosis. (b) AFM force measurements synchronized to the frames (red lines delimitate the frames) (c) Maximum projection x-y, dashed lines delimitate the cantilever bead, pseudo-color scale –F-tractin. Adapted from Nelsen et al., 2019. Courtesy of the authors.
Why do I like this Preprint?
A cell is an active 3D element that can sense and react to mechanical stimuli. The ability to capture dynamic phenomena that happen at small scale is essential to understand cellular mechanics. By using this new method, the prospect of observing cells at high spatiotemporal resolution and directly probing their viscoelastic or adhesive properties open immense possibilities for the field of cell biology and biophysics.
Questions to the authors
You mention that the primary disadvantage of your system is the loss of light collection efficiency when imaging in x-z. Is there a way you could improve this in future versions?
In your previous version (Beicker et al., 2018), you discussed the existence of multiple stiffness regimes in your indentation experiments which is also described by others. Could you in your new system gain some new insights on that? Can your system be of help in calibrating non-Hertzian models? What is your current opinion on the contribution of the nucleus for the viscoelastic behaviour and indentation response?
What is your next step?
Beicker, K., E.T. O’Brien, M.R. Falvo, and R. Superfine. 2018. Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies. Sci. Rep. 8:1504. doi:10.1038/s41598-018-19791-3.
Nelsen, E., C.M. Hobson, M.E. Kern, J.P. Hsiao, E.T. O’Brien, T. Watanabe, B.M. Condon, M. Boyce, S. Grinstein, K.M. Hahn, M.R. Falvo, and R. Superfine. 2019. Combined Atomic Force Microscope and Volumetric Light Sheet System for Mechanobiology. bioRxiv. 812396. doi:10.1101/812396.
Posted on: 12 November 2019 , updated on: 28 November 2019Read preprint
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