Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM.
Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals.
Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.|Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak. E. Sabbelaan 53, 8500 Kortrijk, Belgium.|Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven. Celestijnenlaan 200G box 2403, 3001 Heverlee, Belgium.
Jeroen
J
Vangindertael
Laboratory for Functional Genomics and Proteomics, Division of Animal Physiology and Neurobiology, Department of Biology, KU Leuven. Naamsestraat 59, 3000 Leuven, Belgium.
Isabel
I
Beets
Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.|Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven. Celestijnenlaan 200G box 2403, 3001 Heverlee, Belgium.
Susana
S
Rocha
Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.
Peter
P
Dedecker
Laboratory for Functional Genomics and Proteomics, Division of Animal Physiology and Neurobiology, Department of Biology, KU Leuven. Naamsestraat 59, 3000 Leuven, Belgium.
Liliane
L
Schoofs
Karen
K
Vanhoorelbeke
Laboratory for Thrombosis Research, Interdisciplinary Research Facility Life Sciences, KU Leuven Kulak. E. Sabbelaan 53, 8500 Kortrijk, Belgium.
Karen
K
Vanhoorelbeeke
Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.|Nano-Science Center, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Johan
J
Hofkens
Laboratory for Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven. Celestijnenlaan 200G box 2403, 3001 Heverlee, Belgium.
Hideaki
H
Mizuno
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D016000:Cluster Analysis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000473:pathology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D008164:Luminescent Proteins; D018091:Receptors, AMPA; C096470:glr-1 protein, C elegans