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Supercritical Angle Fluorescence : from microscopy to nanoscopy - Sandrine Leveque-Fort

Institut des Sciences Moléculaires d’Orsay

Supercritical Angle Fluorescence : from microscopy to nanoscopy S. Lévêque-Fort Institut des Sciences Moléculaires d’Orsay (ISMO)

Membrane imaging is of major importance to understand cell motility and adhesion or protein trafficking. Total Internal Reflection Fluorescence microscopy is commonly used to confine the excitation to the basal membrane region. However, it suffers from loss of confinement and inhomogeneous excitation. Supercritical Angle Fluorescence (SAF) emission is an alternative dual depth wide field imaging which takes advantage of evanescent waves at the detection rather than at the excitation,.
The near field components of a fluorophore placed in the vicinity of the glass/cell interface can become propagative and emitted at supercritical angles. This SAF emission appears as a ring beyond the critical angle in the objective back focal plane (BFP). This emission sharply decays with the fluorophore/surface distance z over a characteristic length of about 150 nm. Hence, selecting SAF provides an efficient way to perform wide field axial filtering in dense samples. This optical Fourier filtering in a relay plane of the BFP can be implemented by amplitude or phase modulation of the SAF emission [1,2], and allows one to simultaneously probe intracellular and membrane events.
This intrinsic SAF emission can also be exploited to improve super-resolution technics such as STED or dSTORM/PALM. I will mainly focus on the combination of single molecule localization microscopy with a SAF detection, which provide absolute axial localization of molecule. This 3D absolute method, called “Direct Optical Nanoscopy with Axially Localized Detection” (DONALD), gives an axial localization precision of 15 nm within an axial range of 150 nm above the coverslip, while preserving typical lateral localization precision ( 10 nm) [3]. Axial position can be accessed up to the first 600 nm within the sample, but with lower localization precision. This unique property of absolute axial localization permits a direct combination of axial protein localization which improve 3D co-localization experiments at the nanoscale. In particular the 3D complex architecture of biological structures such as focal adhesion and podosomes can be revealed. Podosomes are adhesion structures involved in the degradation of the extracellular matrix and formed by macrophages and monocyte/macrophage-derived cells. We are investigating how podosomes are organised at the nanoscale, and how this organisation regulates protrusion force generation. I will present the 3D organization of proteins surrounding the F-actin core of podosomes in human macrophages revealed by our DONALD nanoscope

[1] T. Barroca, K. Balaa, J. Delahaye, S. Lévêque-Fort and E. Fort, “Full-field supercritical angle fluorescence microscopy for live cell imaging”, Optics letters 36, 3051 (2011).
[2] T. Barroca, K. Balaa, S. Lévêque-Fort and E. Fort, “Full-field Near-field optical microscope for Cell Imaging”, Phys. Rev. Lett., 108, 218101 (2012).
[3] N. Bourg et al. Direct Optical nanoscopy with axially localized detection, Nature Photon. (2015)


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