Abstract
Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.
Highlights
Structured Illumination Microscopy enables live imaging with sub-diffraction resolution
As they do in the widefield image, the aberrations result in additional intensity structure around the point spread function (PSF) which will affect the signal to noise
The optical aberrations are mathematically described by an expansion of the wavefront in the back pupil plane in Zernike polynomials, which are a complete, orthogonal set of eigenfunctions defined over the unit circle[37]
Summary
Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. To obtain finer detail in complex biological systems, different super-resolution (SR) techniques have been developed to image below the diffraction limit including structured illumination microscopy (SRSIM)[3,4], stimulated emission depletion microscopy (STED)[5,6], photoactivated localization microscopy (PALM)[7,8], and stochastic optical reconstruction microscopy (STORM)[9,10] Among these SR techniques, SIM stands out for its compatibility with live-cell imaging[11]. Li et al.[22] reported an optical-sectioning SIM incorporating direct wavefront sensing AO that achieved fast, high-resolution in vivo structural and functional imaging of neurons in live model animals. All the above-mentioned work combining AO and SIM does not increase the resolution beyond the diffraction limit in the axial direction
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