Nanostructure analysis using spatially modulated illumination microscopy

  title={Nanostructure analysis using spatially modulated illumination microscopy},
  author={David Baddeley and Claudia Batram and Yanina Weiland and Christoph Cremer and Udo Jochen Birk},
  journal={Nature Protocols},
We describe the usage of the spatially modulated illumination (SMI) microscope to estimate the sizes (and/or positions) of fluorescently labeled cellular nanostructures, including a brief introduction to the instrument and its handling. The principle setup of the SMI microscope will be introduced to explain the measures necessary for a successful nanostructure analysis, before the steps for sample preparation, data acquisition and evaluation are given. The protocol starts with cells already… 
Nanosizing by spatially modulated illumination (SMI) microscopy and applications to the nucleus.
This chapter presents the method of spatially modulated illumination (SMI) microscopy, a (far-field) fluorescence microscopy technique featuring structured illumination obtained via a standing wave field laser excitation pattern, a highly valuable tool to access structural parameters of fluorescently labeled macromolecular structures in cells.
SPDM: single molecule superresolution of cellular nanostructures
Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of
SPDM: light microscopy with single-molecule resolution at the nanoscale
Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory.
Spatially modulated illumination microscopy: application perspectives in nuclear nanostructure analysis
A three-dimensional localization precision of less than or equal to 1 nm is expected to become feasible using fluorescence yields typical for single molecule localization microscopy applications, together with its nanosizing capability, this may eventually be used to analyse macromolecular complexes and other nanostructures with a topological resolution, further narrowing the gap to Cryoelectron microscopy.
High-resolution methods for microscopy and nanostructuring of substrate surfaces based on the SIM method (Structured Illumination Microscopy)
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High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy
The present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional SMI data stack to be acquired in less than 2 seconds, and performs live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes.
Analysis of fluorescent nanostructures in biological systems by means of Spectral Position Determination Microscopy (SPDM)
# PM and YW contributed equally to this work. Localization microscopy (LM) has become an established technique that enables effective optical resolutions in the nanometre range. In principle LM is
Superresolution imaging of biological nanostructures by spectral precision distance microscopy
The principles of spectrally assigned localization microscopy (SALM) of biological nanostructures are described, focusing on a special SALM approach, spectral precision distance/position determination microscopy(SPDM) with physically modified fluorochromes (SPDMPhymod), based on high‐precision localization of fluorescent molecules.


Nanosizing of fluorescent objects by spatially modulated illumination microscopy.
With SMI microscopy in combination with SMI VIM calibration, subwavelength object size measurements as small as 40 nm are experimentally feasible with high accuracy.
Spatially modulated illumination microscopy allows axial distance resolution in the nanometer range.
A method of far-field laser fluorescence microscopy is presented to measure relative axial positions of pointlike fluorescent targets and the distance between each target in the range of a few nanometers, allowing topological measurements so far regarded to be beyond the capabilities of light microscopy.
Measuring the size of biological nanostructures with spatially modulated illumination microscopy.
The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting and the hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.
Subwavelength size determination by spatially modulated illumination virtual microscopy.
The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as approximately 20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account.
Nanolocalization measurements in spatially modulated illumination microscopy using two coherent illumination beams
The determination of the 3D nanostructure of specific chromatic regions is highly relevant for an improved understanding of the functional topology of the genome. The use of different spectral
A Dual-Laser, Spatially Modulated Illumination Fluorescence Microscope
To open an avenue into a far field resolution regime far beyond classical limits, techniques of point spread function (PSF) engineering have been developed, e.g., in 4Pior Theta-microscopy, where the full width at half maximum of the 3D PSF, commonly used as the measure of spatial resolution, was modified.
Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations.
A software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope is reported, indicating that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.
Saturated patterned excitation microscopy--a concept for optical resolution improvement.
The theory of nonlinear patterned excitation microscopy is developed for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state and the effects of photon noise are included in the simulations.
Improvement of confocal spectral precision distance microscopy (SPDM)
In recent years, confocal Laser-Scanning microscopy became the most sophisticated microscopic technique for 3D-imaging in biomedical microstructure research. Experimental evidence, however, showed