3D structured illumination microscopy

William M. Dougherty; Paul C. Goodwin

doi:10.1117/12.877595

11 February 2011 3D structured illumination microscopy

William M. Dougherty, Paul C. Goodwin

Proceedings Volume 7905, Single Molecule Spectroscopy and Imaging IV; 790512 (2011) https://doi.org/10.1117/12.877595
Event: SPIE BiOS, 2011, San Francisco, California, United States

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.

Citation Download Citation

William M. Dougherty and Paul C. Goodwin "3D structured illumination microscopy", Proc. SPIE 7905, Single Molecule Spectroscopy and Imaging IV, 790512 (11 February 2011); https://doi.org/10.1117/12.877595

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