Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination

Abstract

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

Structure brings clarity: Structured illumination microscopy in cell biology

“…While in optical sectioning SIM most calculations can be done in real space, the calculations of a HR-SIM image are done in frequency (Fourier) space. Details of the calculation of a HR-SIM image are beyond the scope of this review as recent excellent articles cover these aspects in depth [11,30,31].…”

Light exposure and cell viability in fluorescence microscopy

“…1, 73430 Aalen, Germany. Tel: +49 7361 576 3401; fax: +49 7361 576 3318; e-mail: herbert.schneckenburger@htw-aalen.de structured illumination and subsequent image processing (Neil et al, 1997;Gustafsson et al, 2008). Further resolution (down to about 20−30 nm, and thus far beyond the Abbé limit) was achieved by special techniques, e.g.…”

Quantitative Superresolution Microscopy Reveals Differences in Nuclear DNA Organization of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

“…In short, a periodic illumination pattern results in heterodyne detection of high frequency information that would otherwise be lost. Images are acquired for multiple pattern orientations and phases and computationally recombined as a superresolution image [Gustafsson et al, 2008; Shroff et al, 2009; Wicker et al, 2013]. 3D‐SIM has revealed the subcellular localization of key proteins in cells [Sonnen et al, 2012; Strauss et al, 2012], the fine details of nuclear envelope [Schermelleh et al, 2008], chromosome structure [Carlton, 2008; Flors and Earnshaw, 2011; Green et al, 2011], or even the specialized cellular structure such as endothelial cell fenestrations [Cogger et al, 2010] and the cytokinetic Z ring in live bacteria [Turnbull et al, 2014].…”