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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

PALM of C. elegans VNC.(a,b) Head region of C. elegans with GLR-1 expressing neurons labeled with eGFP (blue) and GLR-1 molecules labeled with mEOS2 (red). eGFP fluorescence was recorded in standard epi-fluorescence mode and only clearly outlines the ventral nerve cord (VNC). Single GLR-1 molecules detected by PALM imaging were plotted as spots with a width of 214 nm to simulate diffraction-limited microscopy. When zooming in (b), GLR-1 puncta are visible in the VNC. (c) PALM image plotted at 25 ± 0.2 nm average resolution ± standard deviation, similar to (b), showing a more detailed structured of the GLR-1 puncta in the VNC. Scale bars measure 1 μm (a) or 250 nm (b,c). (d) Cluster thickness plotted versus cluster length (red dots). The function y = x is shown in blue, the function y = 3x is shown in green. Most clusters are situated between these lines. (e) Cluster thickness plotted as a function of number of molecules per cluster and (f) cluster length plotted as a function of number of molecules per cluster. Distributions shown (e,f) are fitted with a logarithmic function (blue curve). Function and fit coefficients ± s.d. are shown in box.
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f3: PALM of C. elegans VNC.(a,b) Head region of C. elegans with GLR-1 expressing neurons labeled with eGFP (blue) and GLR-1 molecules labeled with mEOS2 (red). eGFP fluorescence was recorded in standard epi-fluorescence mode and only clearly outlines the ventral nerve cord (VNC). Single GLR-1 molecules detected by PALM imaging were plotted as spots with a width of 214 nm to simulate diffraction-limited microscopy. When zooming in (b), GLR-1 puncta are visible in the VNC. (c) PALM image plotted at 25 ± 0.2 nm average resolution ± standard deviation, similar to (b), showing a more detailed structured of the GLR-1 puncta in the VNC. Scale bars measure 1 μm (a) or 250 nm (b,c). (d) Cluster thickness plotted versus cluster length (red dots). The function y = x is shown in blue, the function y = 3x is shown in green. Most clusters are situated between these lines. (e) Cluster thickness plotted as a function of number of molecules per cluster and (f) cluster length plotted as a function of number of molecules per cluster. Distributions shown (e,f) are fitted with a logarithmic function (blue curve). Function and fit coefficients ± s.d. are shown in box.

Mentions: To demonstrate the capability of PALM for nanometre-precise protein localization in intact animals, we visualized the distribution of mEOS2-tagged GLR-1 in the VNC of C. elegans (Fig. 3). GLR-1 is reported to localize in a punctate pattern along these axon bundles2627. We first located the focal plane containing the eGFP labeled VNC by wide-field fluorescence microscopy (Fig. 3 panels a,b and c, blue signals). Subsequent PALM imaging on the VNC yielded the distribution of GLR-1 with an average resolution of 25 nm (Fig. 3c). In addition, we reconstructed images that mimic the diffraction-limited images of GLR-1 puncta by rendering the PALM coordinates with a spot size of 214 nm (equivalent of 2 pixels) (Fig. 3 panels a and b). In these images, we observed puncta with a width of around 800 nm, consistent with the reported size by previous microscopy studies2730. Although there are a larger number of puncta in the VNC, in this particular image only 3 puncta close to the head ganglia were in focus due to the shallow focal depth of the single molecule detection and the orientation of the worm.


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)

PALM of C. elegans VNC.(a,b) Head region of C. elegans with GLR-1 expressing neurons labeled with eGFP (blue) and GLR-1 molecules labeled with mEOS2 (red). eGFP fluorescence was recorded in standard epi-fluorescence mode and only clearly outlines the ventral nerve cord (VNC). Single GLR-1 molecules detected by PALM imaging were plotted as spots with a width of 214 nm to simulate diffraction-limited microscopy. When zooming in (b), GLR-1 puncta are visible in the VNC. (c) PALM image plotted at 25 ± 0.2 nm average resolution ± standard deviation, similar to (b), showing a more detailed structured of the GLR-1 puncta in the VNC. Scale bars measure 1 μm (a) or 250 nm (b,c). (d) Cluster thickness plotted versus cluster length (red dots). The function y = x is shown in blue, the function y = 3x is shown in green. Most clusters are situated between these lines. (e) Cluster thickness plotted as a function of number of molecules per cluster and (f) cluster length plotted as a function of number of molecules per cluster. Distributions shown (e,f) are fitted with a logarithmic function (blue curve). Function and fit coefficients ± s.d. are shown in box.
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Related In: Results  -  Collection

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f3: PALM of C. elegans VNC.(a,b) Head region of C. elegans with GLR-1 expressing neurons labeled with eGFP (blue) and GLR-1 molecules labeled with mEOS2 (red). eGFP fluorescence was recorded in standard epi-fluorescence mode and only clearly outlines the ventral nerve cord (VNC). Single GLR-1 molecules detected by PALM imaging were plotted as spots with a width of 214 nm to simulate diffraction-limited microscopy. When zooming in (b), GLR-1 puncta are visible in the VNC. (c) PALM image plotted at 25 ± 0.2 nm average resolution ± standard deviation, similar to (b), showing a more detailed structured of the GLR-1 puncta in the VNC. Scale bars measure 1 μm (a) or 250 nm (b,c). (d) Cluster thickness plotted versus cluster length (red dots). The function y = x is shown in blue, the function y = 3x is shown in green. Most clusters are situated between these lines. (e) Cluster thickness plotted as a function of number of molecules per cluster and (f) cluster length plotted as a function of number of molecules per cluster. Distributions shown (e,f) are fitted with a logarithmic function (blue curve). Function and fit coefficients ± s.d. are shown in box.
Mentions: To demonstrate the capability of PALM for nanometre-precise protein localization in intact animals, we visualized the distribution of mEOS2-tagged GLR-1 in the VNC of C. elegans (Fig. 3). GLR-1 is reported to localize in a punctate pattern along these axon bundles2627. We first located the focal plane containing the eGFP labeled VNC by wide-field fluorescence microscopy (Fig. 3 panels a,b and c, blue signals). Subsequent PALM imaging on the VNC yielded the distribution of GLR-1 with an average resolution of 25 nm (Fig. 3c). In addition, we reconstructed images that mimic the diffraction-limited images of GLR-1 puncta by rendering the PALM coordinates with a spot size of 214 nm (equivalent of 2 pixels) (Fig. 3 panels a and b). In these images, we observed puncta with a width of around 800 nm, consistent with the reported size by previous microscopy studies2730. Although there are a larger number of puncta in the VNC, in this particular image only 3 puncta close to the head ganglia were in focus due to the shallow focal depth of the single molecule detection and the orientation of the worm.

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