Combining Tip-Scanning AFM with Super-Resolution Optical Imaging towards Multiparametric Correlative Microscopy

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Correlative Microscopy Across the Scales
Dimitar R Stamov (3), Tanja Neumann (3), Ana I Gómez-Varela (1), Adelaide Miranda (2), Pieter A A De Beule (2), Heiko Haschke (3)
1. Department of Applied Physics, University of Santiago de Compostela, Spain
2. International Iberian Nanotechnology Laboratory, Braga, Portugal
3. JPK BioAFM, Bruker Nano GmbH, Berlin, Germany

Atomic Force Microscopy, Multiparametric Correlative Microscopy, high-resolution structural analysis, Topography, Biomechanics

Abstract text

The last three decades have established atomic force microscopy (AFM) as an indispensable tool for high-resolution structural analysis of specimens ranging from single molecules to complex biological systems [1]. AFM currently offers premium spatial resolution of the analysed samples while simultaneously being able to correlate topography and mechanics at near native/physiological imaging conditions. In turn, the combination with advanced/customised optics leverages the advantages of immunolabelling techniques for truly correlative microscopy. Recording the stimulated emission depletion (STED) microscopy fluorescence delivers a multi-colour image with a spatial resolution 6-10 times enhanced relative to conventional optical methods and, therefore, reach the same order of magnitude as the spatial resolution of the AFM [2]. Furthermore, structured illumination microscopy (SIM) offers a unique possibility to go below the optical diffraction limit, while simultaneously operating and acquiring AFM images [3].

We will demonstrate how AFM imaging and super-resolution 2color easy3D STED measurements can be combined and show results on the co-localized imaging and sample manipulation with a precision far below the diffraction limit. This can be applied for comprehensive investigation of biological samples and allow for immunological assignment of the high-resolution cytoskeletal filaments in living fibroblasts. The mechanical stimulation of microtubules and actin filaments with AFM in living cells while performing STED experiments will be presented. We will also show examples of the accuracy of registering/overlay the AFM and optical images on commercially available DNA origami structures with dimensions below the diffraction limit.

Correlating data from different microscopy techniques holds the potential to discover new facets of signalling events in cellular biology. We have recently demonstrated for a first time a hardware set-up capable of achieving simultaneous co-localized imaging of spatially correlated far-field super-resolution fluorescence microscopy and AFM [4]. We will demonstrate the system performance using sub-resolution fluorescent beads, and a test sample consisting of human bone osteosarcoma epithelial cells, with plasma membrane transporter 1 (MCT1).

[1]          M. W. Amrein and D. Stamov, ‘Atomic Force Microscopy in the Life Sciences’, in Springer Handbook of Microscopy, P. W. Hawkes and J. C. H. Spence, 2019, pp. 1469–1505.

[2]          B. Harke et al., Opt. Nanoscopy 1 (1), p. 3, 2012.

[3]          A. Miranda et al., Nanoscale 13 (4), pp. 2082–2099, 2021.

[4]          A. I. Gómez-Varela et al., Sci. Rep. 10 (1), p. 1122, 2020.