Supercritical angle fluorescence for enhanced axial resolution in classical and super-resolution microscopy.

Abstract

Supercritical angle fluorescence (SAF) is an intrinsic component of the emission of any fluorescent molecule. SAF emission corresponds to the near field components of any fluorophore placed in the vicinity of the glass/cell interface which becomes propagative and emitted at supercritical angles. It thus intrinsically presents in the sample, can represent up to 50% of the emission of the fluorophore. This SAF emission sharply decays with the fluorophore/interface distance z over a characteristic length of about 150 nm. We showed that this emission can be used as a sectioning strategy in classical microscopy for live cells imaging, or allow one to localize single molecule event with an absolute 15 nm axial precision in super-resolution microscopy. To decipher SAF emission from undercritcal one, so far physical masks where inserted in the back focal plane of the objective, thus discarding part of the emission which impacts SNR in classical microscopy and localization precision in single molecule localization microscopy (SMLM). This limitation can be overcome by implementing a PSF engineering strategy, thanks to a phase mask which induces a π-shift between SAF and under-critical emissions and allows to benefit of all photons. In classical microscopy, the simultaneous acquisition of an epifluorescence image and a π-shifted SAF, permits real time dual depth imaging of epifluorescence and ϕSAF image, allowing a complete following of receptors trafficking. In combination with SMLM, this permits to detect all emitted photons and thus improve the accessible localization precision. This ϕSAF/SMLM will be presented on various cytoskeleton protein imaging in fixed cells, but also live cells thanks to self blinking dyes.