Abstract
.Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue.Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth.Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices.Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of inside the biological tissue and obtain a 60% signal increase after correction.Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.
Highlights
Introductionstimulated emission depletion (STED) microscopy relies on the use of two co-propagating laser beams: a Gaussian beam to excite fluorescent molecules within a diffraction-limited focal spot and a depletion beam to deexcite the fluorescence of the peripheral molecules via stimulated emission
The advent of super-resolution microscopy, breaking the diffraction barrier of optical microscopy, has opened up tremendous opportunities for nanoscale imaging of live cells.[1,2] In particular, stimulated emission depletion (STED) microscopy has made it possible to reconcile nanoscale resolution with imaging in live brain tissue preparations, setting new standards for visualizing the dynamic morphology of neurons.[3,4,5]STED microscopy relies on the use of two co-propagating laser beams: a Gaussian beam to excite fluorescent molecules within a diffraction-limited focal spot and a depletion beam to deexcite the fluorescence of the peripheral molecules via stimulated emission
After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices
Summary
STED microscopy relies on the use of two co-propagating laser beams: a Gaussian beam to excite fluorescent molecules within a diffraction-limited focal spot and a depletion beam to deexcite the fluorescence of the peripheral molecules via stimulated emission. Increases the spatial resolution by up to an order of magnitude.[6] Using appropriate beam shaping, STED microscopy can be operated either in 2D or 3D mode, typically using so-called donut or bottle beams Applied in combination, these two modes allow for optimizing lateral as well as axial resolution at the same time.[7,8,9]
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