Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

Eva Wegel,B Christoffer Lagerholm,Alan Wainman,Rainer Kaufmann,Ian M Dobbie,Stephan Uphoff,Antonia Göhler

doi:10.1038/srep27290

Abstract

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

Highlights

The resolution of the light microscope is limited by Abbe’s Law to 200–250 nm in the lateral and 500– 700 nm in the axial direction
In STimulated Emission Depletion (STED) a diffraction-limited spot is excited at one wavelength while a super-imposed, red-shifted, second laser beam, projected to a donut-shape, depletes almost all emission laterally leaving only a central focal spot with a dimension below the diffraction limit[10,11]
For the super-resolution comparisons we chose a range of biological structures that vary in shape and complexity in all three dimensions, i.e. strongly fluorescent 25 nm filaments of varying density in the cell, 9 nm filaments in two close layers of fine meshwork, hollow barrels, vesicles and tubules in a complicated 3D arrangement

Summary

Introduction

The resolution of the light microscope is limited by Abbe’s Law to 200–250 nm in the lateral and 500– 700 nm in the axial direction. The most commonly used super-resolution techniques are STED, SIM and SMLM all of which have been commercialized in the last few years (for detailed reviews of the different techniques see[1,2,3]). In Structured Illumination Microscopy (SIM)[4,5,6] an adapted wide-field microscope setup uses patterned illumination, usually stripes, to excite the sample. More recent development is time-gated STED (gSTED), which utilises pulsed excitation combined with continuous wave (CW) laser depletion and time-gated detection[13]. By taking a few thousand to tens of thousands of images each with a different subset of fluorescent molecules, the position information of the fluorophores can be used to reconstruct an image with a resolution that mainly depends on the number of detected photons[14]. Commercial solutions are available from Leica (Leica GSD), Nikon (N-STORM), Zeiss (Zeiss Elyra P1) and Bruker (Vutara 350)

Methods

Results

Conclusion