Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Pereira, A. J.; Sousa, M.; Almeida, A. C.; Ferreira, L. T.; Costa, A. R.; Novais-Cruz, M.; Ferras, C.; Sousa, M. M.; Sampaio, P.; Belsley, M.; Maiato, H.

doi:10.1101/381343

bioRxiv · 10.1101/381343

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Pereira, A. J.·Sousa, M.·Almeida, A. C.·Ferreira, L. T.·Costa, A. R.·Novais-Cruz, M.·Ferras, C.·Sousa, M. M.·Sampaio, P.·Belsley, M.·Maiato, H.

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Abstract

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

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Pereira, A. J., Sousa, M., Almeida, A. C., Ferreira, L. T., Costa, A. R., Novais-Cruz, M., Ferras, C., Sousa, M. M., Sampaio, P., Belsley, M., Maiato, H.. 2018-07-31. Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast. https://doi.org/10.1101/381343

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