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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

Single molecule detection in C. elegans.(a,b) Raw images (10 by 10 pixels) data showing 2 randomly chosen single molecules inside C. elegans. (c,d) 3D representation of the single molecule signal (top), 2D Gaussian fit (middle) and residuals after fitting (bottom) shown in (a) and (b) respectively. The y-axes on the 3D-plots denote the intensity in arbitrary units (e,f) Intensity time trace of the molecules shown in (a,b) indicating that mEOS2 blinks on and off multiple times, leading to possible multiple detections. (g) Graph showing the amount of detected unique molecules in function of the allowed dark time (td) between detections (red circles). Exponential fit (blue line) shows the onset of a plateau at around 2 seconds.
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f2: Single molecule detection in C. elegans.(a,b) Raw images (10 by 10 pixels) data showing 2 randomly chosen single molecules inside C. elegans. (c,d) 3D representation of the single molecule signal (top), 2D Gaussian fit (middle) and residuals after fitting (bottom) shown in (a) and (b) respectively. The y-axes on the 3D-plots denote the intensity in arbitrary units (e,f) Intensity time trace of the molecules shown in (a,b) indicating that mEOS2 blinks on and off multiple times, leading to possible multiple detections. (g) Graph showing the amount of detected unique molecules in function of the allowed dark time (td) between detections (red circles). Exponential fit (blue line) shows the onset of a plateau at around 2 seconds.

Mentions: To visualize the single molecule distribution of GLR-1, we subjected the mounted C. elegans to PALM imaging. Thanks to the restricted expression of mEOS2-labeled GLR-1 in only a small subset of neurons, background fluorescence was suppressed enough to detect respective single molecules allowing for PALM. We were able to detect single molecules under Köhler illumination up to approximately 10 μm deep (nominal focus position)35, although the background was reduced even further under highly inclined and laminated optical sheet (HILO) illumination36. We then sought to validate the quality of the recorded single molecule signals (Supplementary movie 2). The intensity-to-noise ratios of the detected molecules were mostly in the range of 6 to 8, allowing us to calculate their coordinates with an average accuracy of 20–30 nm13. After 2D Gaussian fitting using the Localizer software package37, over 24,000 spots could be detected in a representative PALM data set of a single worm (Fig. 2). Further analysis yielded the coordinates of these single molecules with an average accuracy of 25 nm (Fig. 2 panels a, b, c and d; Supplementary figure 3).


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)

Single molecule detection in C. elegans.(a,b) Raw images (10 by 10 pixels) data showing 2 randomly chosen single molecules inside C. elegans. (c,d) 3D representation of the single molecule signal (top), 2D Gaussian fit (middle) and residuals after fitting (bottom) shown in (a) and (b) respectively. The y-axes on the 3D-plots denote the intensity in arbitrary units (e,f) Intensity time trace of the molecules shown in (a,b) indicating that mEOS2 blinks on and off multiple times, leading to possible multiple detections. (g) Graph showing the amount of detected unique molecules in function of the allowed dark time (td) between detections (red circles). Exponential fit (blue line) shows the onset of a plateau at around 2 seconds.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555104&req=5

f2: Single molecule detection in C. elegans.(a,b) Raw images (10 by 10 pixels) data showing 2 randomly chosen single molecules inside C. elegans. (c,d) 3D representation of the single molecule signal (top), 2D Gaussian fit (middle) and residuals after fitting (bottom) shown in (a) and (b) respectively. The y-axes on the 3D-plots denote the intensity in arbitrary units (e,f) Intensity time trace of the molecules shown in (a,b) indicating that mEOS2 blinks on and off multiple times, leading to possible multiple detections. (g) Graph showing the amount of detected unique molecules in function of the allowed dark time (td) between detections (red circles). Exponential fit (blue line) shows the onset of a plateau at around 2 seconds.
Mentions: To visualize the single molecule distribution of GLR-1, we subjected the mounted C. elegans to PALM imaging. Thanks to the restricted expression of mEOS2-labeled GLR-1 in only a small subset of neurons, background fluorescence was suppressed enough to detect respective single molecules allowing for PALM. We were able to detect single molecules under Köhler illumination up to approximately 10 μm deep (nominal focus position)35, although the background was reduced even further under highly inclined and laminated optical sheet (HILO) illumination36. We then sought to validate the quality of the recorded single molecule signals (Supplementary movie 2). The intensity-to-noise ratios of the detected molecules were mostly in the range of 6 to 8, allowing us to calculate their coordinates with an average accuracy of 20–30 nm13. After 2D Gaussian fitting using the Localizer software package37, over 24,000 spots could be detected in a representative PALM data set of a single worm (Fig. 2). Further analysis yielded the coordinates of these single molecules with an average accuracy of 25 nm (Fig. 2 panels a, b, c and d; Supplementary figure 3).

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