Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination (original) (raw)
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Image formation in structured illumination wide-field fluorescence microscopy
Micron, 2008
We present a theoretical analysis of the image formation in structured illumination wide-field fluorescence microscopy (SIWFFM). We show that the optically sectioned images obtained with this approach possess the optical sectioning strengths comparable to those obtained with the confocal microscope. We further show that the transfer function behaviour is directly comparable to that of the true confocal instrument. The theoretical considerations are compared with and confirmed by experimental results.
Structured illumination microscopy of a living cell
European Biophysics Journal, 2009
Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells.
Structured illumination microscopy for super-resolution and optical sectioning
Chinese Science Bulletin, 2014
Optical microscopy plays an essential role in biological studies due to its capability and compatibility of non-contact, minimally invasive observation and measurement of live specimens. However, the conventional optical microscopy only has a spatial resolution about 200 nm due to the Abbe diffraction limit, and also lacks the ability of three-dimensional imaging. Super-resolution farfield optical microscopy based on special illumination schemes has been dramatically developed over the last decade. Among them, only the structured illumination microscopy (SIM) is of wide-field geometry that enables it easily compatible with the conventional optical microscope. In this article, the principle of SIM was introduced in terms of point spread function (PSF) and optical transform function (OTF) of the optical system. The SIM for super-resolution (SIM-SR) proposed by Gustafsson et al. and the SIM for optical sectioning (SIM-OS) proposed by Neil et al. are the most popular ones in the research community of microscopy. They have the same optical configuration, but with different data postprocessing algorithms. We mathematically described the basic theories for both of the SIMs, respectively, and presented some numerical simulations to show the effects of super-resolution and optical sectioning. Various approaches to generation of structured illumination patterns were reviewed. As an example, a SIM system based on DMDmodulation and LED-illumination was demonstrated. A lateral resolution of 90 nm was achieved with gold nanoparticles. The optical sectioning capability of the microscope was demonstrated with Golgi-stained mouse brain neurons, and the sectioning strength of 930 nm was obtained.
I5S: Wide-Field Light Microscopy with 100-nm-Scale Resolution in Three Dimensions
Biophysical Journal, 2008
A new type of wide-field fluorescence microscopy is described, which produces 100-nm-scale spatial resolution in all three dimensions, by using structured illumination in a microscope that has two opposing objective lenses. Illumination light is split by a grating and a beam splitter into six mutually coherent beams, three of which enter the specimen through each objective lens. The resulting illumination intensity pattern contains high spatial frequency components both axially and laterally. In addition, the emission is collected by both objective lenses coherently, and combined interferometrically on a single camera, resulting in a detection transfer function with axially extended support. These two effects combine to produce near-isotropic resolution. Experimental images of test samples and biological specimens confirm the theoretical predictions.
Widefield fluorescence microscopy with extended resolution
Histochemistry and Cell Biology, 2008
Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.
Nature Methods, 2012
We demonstrate 3D super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) let us physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples 8-fold thicker than previously demonstrated with SIM. We imaged a variety of samples at one 2D image per second, at resolutions down to 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths greater than 45 μm. We also captured dynamic changes in the zebrafish lateral line primordium and observed the interactions between myosin IIA and F-actin in cells encapsulated within collagen gels, obtaining two-color 4D super-resolution datasets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with widefield microscopes.
Resolution in structured illumination microscopy: a probabilistic approach
Journal of the Optical Society of America A, 2008
Structured illumination can be employed to extend the lateral resolution of wide-field fluorescence microscopy. Since a structured illumination microscopy image is reconstructed from a series of several acquired images, we develop a modified formulation of the imaging response of the microscope and a probabilistic analysis to assess the resolution performance. We use this model to compare the fluorescence imaging performance of structured illumination techniques to confocal microscopy. Specifically, we examine the trade-off between achievable lateral resolution and signal-to-noise ratio when photon shot noise is dominant. We conclude that for a given photon budget, structured illumination invariably achieves better lateral resolution than confocal microscopy.
Proceedings of the National Academy of Sciences of the United States of America, 2017
Light-sheet-based fluorescence microscopy (LSFM) features optical sectioning in the excitation process. It minimizes fluorophore bleaching as well as phototoxic effects and provides a true axial resolution. The detection path resembles properties of conventional fluorescence microscopy. Structured illumination microscopy (SIM) is attractive for superresolution because of its moderate excitation intensity, high acquisition speed, and compatibility with all fluorophores. We introduce SIM to LSFM because the combination pushes the lateral resolution to the physical limit of linear SIM. The instrument requires three objective lenses and relies on methods to control two counterpropagating coherent light sheets that generate excitation patterns in the focal plane of the detection lens. SIM patterns with the finest line spacing in the far field become available along multiple orientations. Flexible control of rotation, frequency, and phase shift of the perfectly modulated light sheet are d...