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Forschungsarbeit

Breaking the Diffraction Limit in Microscopy with Structured

Illumination

By Florian Ströhl (20.01.2015)

One of the most important tools for biomedical research is fluorescence microscopy. The method of selective molecule labelling enables the observation of process kinetics within living cells – conventional wide-field microscopes (WF), however, are limited to a maximum resolution of half the wavelength in use due to optical diffraction. It was discovered that it is possible to further increase resolution by using structured instead of homogeneous illumination light fields; the particular illumination structure, in turn, crucially affects the achievable image sharpness and the overall performance of the so-called superresolution microscopes.

In structured illumination microscopy (SIM) a boost in resolution is achieved by spatial frequency mixing of illumination and fluorophore distribution [1]. This is a phenomenon known to physicists as Moiré effect. The very same effect also causes stripy patterns to appear on lumberjack shirts on television – here the pattern of the shirt and the pixelated structure of the TV mix. In SIM the Moiré effect encodes the extremely fine details of the sample structure, which are usually unobservable, in the observable Moiré patterns. The thus obtained additional information is computationally decoded afterwards to offer super-resolution. However, depending on the particular structure of the illumination light, the respective reconstruction algorithms and imaging performances vary a lot. In addition, the set-ups and acquisition procedures need to be adapted to generate the needed patterns and record the appropriate amount of raw data files. Therefore it is useful to compare different illumination geometries as they all have their strengths, weaknesses and optimal fields of application.

Ströhl: Fig. 1[Bildunterschrift / Subline]: Figure 1: A comparison of different SIM modalities to conventional wide-field microscopy. (a) shows the resolution improvement of MSIM on red blood cells – a thick sample; (b) and (c) show the performance of OS-SIM and SROS-SIM on fluorescence nano-spheres, which are smaller than the diffraction limit of light. (Figure modified from [2])

For example, sinusoidal illumination in a total internal reflection configuration (TIRF-SIM) allows extremely fast, high-contrast imaging with the theoretical possibility of unlimited resolution improvement via the use of non-linear effects (SSIM) but is restrained to two-dimensional imaging [3]. In contrast, three-beam sinusoidal illumination (3D-SIM) requires more reconstruction and hardware efforts but is capable of removing out-of-focus light computationally whilst increasing resolution isotropically in all three dimensions, thus producing volumetric super-resolution data-sets. A derivation of this approach is super-resolution optical sectioning SIM (SROS-SIM). It also removes out-of-focus light computationally and enables volumetric data at even higher imaging rates than 3D-SIM but lacks resolution improvement in the axial direction. OS-SIM, another derivation, is capable of enhancing contrast and optical sectioning – its main advantage is the comparably easy reconstruction procedure. Diffraction limited multi-focal spot pattern illumination, MSIM, is similar to SROS-SIM and offers super-resolution in the lateral direction. Although it has slower imaging rates it enhances penetration depth and contrast and is, therefore, useful in thick sample imaging [2]. It must be mentioned that there exist a multitude of other super-resolution techniques, all optimally suitable for different fields of application [4]. Therefore, a lot of work is waiting.


References:

[1] Heintzmann R and Cremer C G (1999), Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating, Proc. SPIE 3568, 185-196

[2] Ströhl F and Kaminski C F (2015), A Joint Richardson-Lucy Deconvolution Algorithm for the Reconstruction of Multifocal Structured Illumination Microscopy Data, Methods Appl. Fluoresc. 3 014002

[3] Gustafsson M G L (2005), Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA. 102 37 13081–13086

[4] Schermelleh L, Heintzmann R, Leonhardt H (2010), A guide to super‐resolution fluorescence microscopy, J. Cell Biol. 190 165–175


Scientific Career
  • 2009-2012
  • B.Sc. in Medical Engineering, University of Erlangen-Nuremberg
  • 2012-2014
  • M.Sc. (hons) in Advanced Optical Technologies, University of Erlangen-Nuremberg
  • Since 2014
  • PhD in Biotechnology, University of Cambridge, UK

Scholarships and Awards
  • * DPG award for outstanding achievements in high-school physics (2009)
  • * Stipendiary of the Studienstiftung des deutschen Volkes (2011)
  • * E-fellows.net online studentship (2012)
  • * Member of FAU’s Leonardo-Kolleg (2013)
  • * BMBF award for outstanding achievements in medical engineering studies (2014)
Publications
  • * Ströhl F and Kaminski C F (2015), A Joint Richardson-Lucy Deconvolution Algorithm for the Reconstruction of Multifocal Structured Illumination Microscopy Data, Methods Appl. Fluoresc. 3 014002