Super-resolution imaging of the bacterial cytoskeleton

Introduction

High resolution fluorescence microscopy is of special interest in all life sciences as it allows to investigate living matter like e.g. cells. In combination with structured illumination microscopy (SIM) one can increase the lateral resolution of fluorescence microscopes beyond the classical Rayleigh resolution limit (≈ 250 nm) to about 100nm. Applying this to biological samples enables the revelation of otherwise undetectable details, even in living cells. We combine an objective launched total internal reflection fluorescence (TIRF) set-up with structured illumination. This brings together the advantages of high contrast and a superior resolution. The evanescent sinusoidal light grid is created with the help of a spatial light modulator (SLM). In combination with a fast camera for image acquisition this enables high frame rates of up to 1 Hz, sufficient to image many dynamic processes in living cells. The combination of TIRF-microscopy with structured illumination (TIRF-SIM) yields images of high contrast with superresolution of biological samples. We image strongly scattering cells as well as living cells that show dynamics on the timescales of seconds with about two fold increased resolution. Finer structures, relevant for the biological understanding of the cells, can be discriminated. Our images prove the outstanding capabilities of TIRF-SIM for live cell imaging.

Technique

Figure 1: Generation of structured evanescent illumination by interference of two plane waves

We use an objective-launched TIRF-setup with a high-NA objective lens. A laser beam incident on a spatial light modulator (SLM) is diffracted into two beams. These are focused by the objective so that they enter the focal plane under a high angle resulting in total internal reflection at the glass-water-interface. Thus two evanescent waves are created forming a standing wave, which in turn is the illumination pattern. From several images taken with different orientations and phases of the illumination pattern one can computationally reconstruct an image with higher resolution (superresolution).

Figure 2: TIRF-SIM microscope setup

Results

Figure 3: Superresolution: Difference between a normal TIRF image and a reconstructed TIRF-SIM image of 92 nm fluorescent beads. The increase in resolution is apparent as much finer details can be resolved: In some clusters the single beads can only be resolved in the TIRF-SIM image.

Figure 4: Yeast cell with the fluorescently labeled membrane protein Sag1. (Left) Normal TIRF image. (Right) TIRF-SIM image of the same cell showing higer resolution and contrast. Scale bar is 1 µm. (Collaboration with the lab of R. Wedlich-Söldner)

Figure 5: MreB dynamics in the bacteria Bacillus Subtilis. (Left) Brightfield image of a B. subtilis cell. (Right) TIRF-SIM time series of GFP-labeled MreB in the same cell showing high dynamics. Scale bar is 1 µm.
(Collaboration with the lab of P. Graumann)

People involved in this project:
>>> Thomas Gerrer

This project is supported by

Collaborations:

• Prof. Peter Graumann - SYNMIKRO - University of Marburg
  & H. Joel D. Soufo - Microbiology - University of Freiburg
• Prof. Roland Wedlich-Söldner - MPI of Biochemistry - Martinsried

Relevant Publications:

[1] Olshausen P, Rohrbach A
Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit
2013 Opt Lett, Band: 38, Nummer: 20, Seiten: 4066 - 4069

[2] Olshausen P, Soufo H, Wicker K, Heintzmann R, Graumann P, Rohrbach A
Superresolution Imaging of Dynamic MreB Filaments in B. subtilis - A Multiple-Motor-Driven Transport?
2013 Biophys J, Band: 105, Nummer: 5, Seiten: 1171 - 1181

[3] F. Spira, N. S. Mueller, G. Beck, P. von Olshausen, J. Beig and R. Wedlich-Söldner, Patchwork organization of the yeast plasma membrane into numerous coexisting domains, Nature Cell Biology, Vol. 14, pp. 1-11, (2012)

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