Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.
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.
Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Eva
E
Wegel
Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Antonia
A
Göhler
Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
B Christoffer
BC
Lagerholm
Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.|Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Alan
A
Wainman
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Stephan
S
Uphoff
Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.|Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
Rainer
R
Kaufmann
Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Ian M
IM
Dobbie
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008264:Macrophages / Q000166:cytology*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure*; D061848:Optical Imaging / Q000379:methods*; D000072760:Single Molecule Imaging