High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

  title={High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy},
  author={J{\"u}rgen Reymann and David Baddeley and Manuel Gunkel and Paul Lemmer and Werner Stadter and Thibaud Jegou and Karsten Rippe and Christoph Cremer and Udo Jochen Birk},
  journal={Chromosome Research},
Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico… 
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.
Application perspectives of localization microscopy in virology
Additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligonucleotide fluorescence in situ hybridization, as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices are discussed.
Measurement of replication structures at the nanometer scale using super-resolution light microscopy
These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures
A special method of localisation microscopy, spectral precision distance/position determination microscopy and its combination with fluorescence in situ hybridization is reported on to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level.
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
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.
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.


Nanostructure of specific chromatin regions and nuclear complexes
The application of SMI microscopy to size measurements of the 7q22 gene region is presented, giving a size estimate of 105±16 nm which corresponds to an average compaction ratio of 1:324, demonstrating that the technique is robust when applied to biological samples.
Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast
Three-dimensional imaging of live eukaryotic cells at an equilateral resolution of ≈100 nm is demonstrated for the first time, and mitochondria of cells grown on medium containing glycerol as the only carbon source, as opposed to glucose-grown cells, exhibit a strongly branched tubular reticulum.
Macromolecular-scale resolution in biological fluorescence microscopy.
Far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples is demonstrated, enabling an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes.
Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy
Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved
Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy.
The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei and indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.
Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation
A fluorescence microscope in which axial resolution is increased to better than 0.05 μm by using the principle of standing-wave excitation of fluorescence to obtain images of actin fibres and Filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.
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.
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.
Nanostructure analysis using spatially modulated illumination microscopy
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.
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.