11 February 2011 3D structured illumination microscopy
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Abstract
Three-dimensional structured illumination microscopy achieves double the lateral and axial resolution of wide-field microscopy, using conventional fluorescent dyes, proteins and sample preparation techniques. A three-dimensional interference-fringe pattern excites the fluorescence, filling in the "missing cone" of the wide field optical transfer function, thereby enabling axial (z) discrimination. The pattern acts as a spatial carrier frequency that mixes with the higher spatial frequency components of the image, which usually succumb to the diffraction limit. The fluorescence image encodes the high frequency content as a down-mixed, moiré-like pattern. A series of images is required, wherein the 3D pattern is shifted and rotated, providing down-mixed data for a system of linear equations. Super-resolution is obtained by solving these equations. The speed with which the image series can be obtained can be a problem for the microscopy of living cells. Challenges include pattern-switching speeds, optical efficiency, wavefront quality and fringe contrast, fringe pitch optimization, and polarization issues. We will review some recent developments in 3D-SIM hardware with the goal of super-resolved z-stacks of motile cells.
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William M. Dougherty, William M. Dougherty, Paul C. Goodwin, Paul C. Goodwin, } "3D structured illumination microscopy", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790512 (11 February 2011); doi: 10.1117/12.877595; https://doi.org/10.1117/12.877595
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