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Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM.

Vangindertael J, Beets I, Rocha S, Dedecker P, Schoofs L, Vanhoorelbeeke K, Hofkens J, Mizuno H - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Confocal correlated PALM in C. elegans.(a) Partial confocal Z-projection (15 optical slices, total thickness of 5.30 μm) of eGFP fluorescence marking GLR-1 positive neurons and their processes (blue) overlaid with transmission image of the C. elegans head region (grey). White arrowheads indicate the nerve ring and the VNC. (b) Enlargement of GLR-1 expressing head neurons from Z-projection in (a). (c,d) Close up of the AVEL neuron (c) and the nerve ring (d) indicated in (b) with the distribution of GLR-1 mapped onto the neuron by ccPALM. (e,f) Close up of box in panels (c) and (d), respectively. Scale bars indicate 2 μm (a,b) 250 nm (c,d), or 100 nm (e,f). Cut-off resolution of PALM images (c–f) is 20 nm.
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f4: Confocal correlated PALM in C. elegans.(a) Partial confocal Z-projection (15 optical slices, total thickness of 5.30 μm) of eGFP fluorescence marking GLR-1 positive neurons and their processes (blue) overlaid with transmission image of the C. elegans head region (grey). White arrowheads indicate the nerve ring and the VNC. (b) Enlargement of GLR-1 expressing head neurons from Z-projection in (a). (c,d) Close up of the AVEL neuron (c) and the nerve ring (d) indicated in (b) with the distribution of GLR-1 mapped onto the neuron by ccPALM. (e,f) Close up of box in panels (c) and (d), respectively. Scale bars indicate 2 μm (a,b) 250 nm (c,d), or 100 nm (e,f). Cut-off resolution of PALM images (c–f) is 20 nm.

Mentions: In addition to the GLR-1 distribution in axon bundles of the VNC, we aimed to determine the super-resolution pattern of GLR-1 in the densely packed neuronal head ganglia and the nerve ring (Fig. 1a). Due to the complex three-dimensional organization of neurons in these head regions, wide-field fluorescence microscopy did not suffice to distinguish individual eGFP labeled neurites and their cell bodies. As a consequence of the small depth-of-field typical for a high-magnification objective, out-of-focus fluorescence blurred the image (Fig. 3a). Confocal microscopy was specifically developed to circumvent this problem and is the standard imaging technique for thick fluorescently labeled biological specimens44. Therefore, we integrated PALM with confocal microscopy in an attempt to correlate the three-dimensional overview of GLR-1 expressing cells with the super-resolution pattern obtained by PALM. For this purpose, we first acquired a confocal Z-stack of the C. elegans head region, and annotated neurons that express GLR-1. The confocal image allowed us to adjust the focus to a focal plane-of-interest on which we subsequently performed PALM imaging. After analysis of the PALM dataset, we overlaid the single molecule distribution map of GLR-1 with the confocal image, thereby correlating the super-resolution data with its anatomical context (Fig. 4). For the left AVE interneuron (AVEL), we observed clusters of GLR-1 restricted to the outer edge of the cell soma that likely denotes the position of the plasma membrane (Fig. 4 panels c and e). We observed similar distributions for GLR-1 in other neurons, e.g. RIMR (Supplementary figure 7), and saw comparable clusters distributed throughout the nerve ring, where multiple synapses are expected to be present21 (Fig. 4 panels d and f).


Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM.

Vangindertael J, Beets I, Rocha S, Dedecker P, Schoofs L, Vanhoorelbeeke K, Hofkens J, Mizuno H - Sci Rep (2015)

Confocal correlated PALM in C. elegans.(a) Partial confocal Z-projection (15 optical slices, total thickness of 5.30 μm) of eGFP fluorescence marking GLR-1 positive neurons and their processes (blue) overlaid with transmission image of the C. elegans head region (grey). White arrowheads indicate the nerve ring and the VNC. (b) Enlargement of GLR-1 expressing head neurons from Z-projection in (a). (c,d) Close up of the AVEL neuron (c) and the nerve ring (d) indicated in (b) with the distribution of GLR-1 mapped onto the neuron by ccPALM. (e,f) Close up of box in panels (c) and (d), respectively. Scale bars indicate 2 μm (a,b) 250 nm (c,d), or 100 nm (e,f). Cut-off resolution of PALM images (c–f) is 20 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4555104&req=5

f4: Confocal correlated PALM in C. elegans.(a) Partial confocal Z-projection (15 optical slices, total thickness of 5.30 μm) of eGFP fluorescence marking GLR-1 positive neurons and their processes (blue) overlaid with transmission image of the C. elegans head region (grey). White arrowheads indicate the nerve ring and the VNC. (b) Enlargement of GLR-1 expressing head neurons from Z-projection in (a). (c,d) Close up of the AVEL neuron (c) and the nerve ring (d) indicated in (b) with the distribution of GLR-1 mapped onto the neuron by ccPALM. (e,f) Close up of box in panels (c) and (d), respectively. Scale bars indicate 2 μm (a,b) 250 nm (c,d), or 100 nm (e,f). Cut-off resolution of PALM images (c–f) is 20 nm.
Mentions: In addition to the GLR-1 distribution in axon bundles of the VNC, we aimed to determine the super-resolution pattern of GLR-1 in the densely packed neuronal head ganglia and the nerve ring (Fig. 1a). Due to the complex three-dimensional organization of neurons in these head regions, wide-field fluorescence microscopy did not suffice to distinguish individual eGFP labeled neurites and their cell bodies. As a consequence of the small depth-of-field typical for a high-magnification objective, out-of-focus fluorescence blurred the image (Fig. 3a). Confocal microscopy was specifically developed to circumvent this problem and is the standard imaging technique for thick fluorescently labeled biological specimens44. Therefore, we integrated PALM with confocal microscopy in an attempt to correlate the three-dimensional overview of GLR-1 expressing cells with the super-resolution pattern obtained by PALM. For this purpose, we first acquired a confocal Z-stack of the C. elegans head region, and annotated neurons that express GLR-1. The confocal image allowed us to adjust the focus to a focal plane-of-interest on which we subsequently performed PALM imaging. After analysis of the PALM dataset, we overlaid the single molecule distribution map of GLR-1 with the confocal image, thereby correlating the super-resolution data with its anatomical context (Fig. 4). For the left AVE interneuron (AVEL), we observed clusters of GLR-1 restricted to the outer edge of the cell soma that likely denotes the position of the plasma membrane (Fig. 4 panels c and e). We observed similar distributions for GLR-1 in other neurons, e.g. RIMR (Supplementary figure 7), and saw comparable clusters distributed throughout the nerve ring, where multiple synapses are expected to be present21 (Fig. 4 panels d and f).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Photochemistry and Spectroscopy, Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven. Celestijnenlaan 200F, 3001 Heverlee, Belgium.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus