@inbook {47131, title = {Structured illumination microscopy (Chapter 15.)}, booktitle = {Fluorescence Microscopy: Super-resolution and other novel techniques}, year = {2014}, pages = {213{\textendash}225}, doi = {10.1016/B978-0-12-409513-7.00015-4}, author = {Shao, Lin and Rego, E Hesper} } @article {4489, title = {Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Jan 17}, pages = {E135-43}, abstract = {

Using ultralow light intensities that are well suited for investigating biological samples, we demonstrate whole-cell superresolution imaging by nonlinear structured-illumination microscopy. Structured-illumination microscopy can increase the spatial resolution of a wide-field light microscope by a factor of two, with greater resolution extension possible if the emission rate of the sample responds nonlinearly to the illumination intensity. Saturating the fluorophore excited state is one such nonlinear response, and a realization of this idea, saturated structured-illumination microscopy, has achieved approximately 50-nm resolution on dye-filled polystyrene beads. Unfortunately, because saturation requires extremely high light intensities that are likely to accelerate photobleaching and damage even fixed tissue, this implementation is of limited use for studying biological samples. Here, reversible photoswitching of a fluorescent protein provides the required nonlinearity at light intensities six orders of magnitude lower than those needed for saturation. We experimentally demonstrate approximately 40-nm resolution on purified microtubules labeled with the fluorescent photoswitchable protein Dronpa, and we visualize cellular structures by imaging the mammalian nuclear pore and actin cytoskeleton. As a result, nonlinear structured-illumination microscopy is now a biologically compatible superresolution imaging method.

}, issn = {1091-6490}, doi = {10.1073/pnas.1107547108}, author = {Rego, E Hesper and Shao, Lin and Macklin, John J and Winoto, Lukman and Johansson, G{\"o}ran A and Kamps-Hughes, Nicholas and Davidson, Michael W and Gustafsson, Mats G L} } @article {4632, title = {Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, year = {2012}, month = {2012 Apr 3}, pages = {5311-5}, abstract = {

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

}, issn = {1091-6490}, doi = {10.1073/pnas.1119262109}, author = {Fiolka, Reto and Shao, Lin and Rego, E Hesper and Davidson, Michael W and Gustafsson, Mats G L} } @article {5309, title = {Super-resolution 3D microscopy of live whole cells using structured illumination.}, journal = {Nature Methods}, volume = {8}, year = {2011}, month = {2011 Dec}, pages = {1044-6}, abstract = {

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for \>50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.

}, keywords = {Animals, Cell Line, Cell Survival, Drosophila melanogaster, Fluorescence, Hela Cells, Humans, Imaging, Microscopy, Microtubules, Mitochondria, Three-Dimensional}, issn = {1548-7105}, doi = {10.1038/nmeth.1734}, author = {Shao, Lin and Kner, Peter and Rego, E Hesper and Gustafsson, Mats G L} }