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Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.

Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, Zimmer C, Dahan M - PLoS ONE (2011)

Bottom Line: Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute.Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes.By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Kastler Brossel, CNRS UMR 8552, Department of Physics, École Normale Supérieure, Université Pierre et Marie Curie-Paris 6, Paris, France.

ABSTRACT
The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

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Related in: MedlinePlus

Super-resolution dynamics: spatial versus temporal resolution.(A) Optimization of the SNR as a function of the image acquisition time. The left panels show the histograms of the amplitudes of the recorded signals at different acquisition times (12.5 ms to 500 ms); the right panels show their respective SNRs. The mean values are indicated as red lines. (B) PALM reconstruction of fixed dendritic spines from 500, 1000, 2000, and 5000 frames of 25 ms. (C) Standard deviation of the distribution of signals across a thin filopodium from the live recording shown in Fig. S2, as a function of the time window used for image reconstruction. (D) Time course of the number of detected molecules for ABP-tdEosFP and actin-PAmCherry at constant illumination. The arrow indicates an increase of the photoconversion laser power at the end of the experiment (5× for ABP-tdEosFP, 10× for actin-PAmCherry).
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pone-0015611-g003: Super-resolution dynamics: spatial versus temporal resolution.(A) Optimization of the SNR as a function of the image acquisition time. The left panels show the histograms of the amplitudes of the recorded signals at different acquisition times (12.5 ms to 500 ms); the right panels show their respective SNRs. The mean values are indicated as red lines. (B) PALM reconstruction of fixed dendritic spines from 500, 1000, 2000, and 5000 frames of 25 ms. (C) Standard deviation of the distribution of signals across a thin filopodium from the live recording shown in Fig. S2, as a function of the time window used for image reconstruction. (D) Time course of the number of detected molecules for ABP-tdEosFP and actin-PAmCherry at constant illumination. The arrow indicates an increase of the photoconversion laser power at the end of the experiment (5× for ABP-tdEosFP, 10× for actin-PAmCherry).

Mentions: In order to determine the best conditions for live PALM imaging, we adjusted the image acquisition time to optimize the signal-to-noise-ratio (SNR) of the individual fluorophores. To ensure the fastest possible sampling of the structures, we chose the maximal excitation laser power (561 nm at 4 kW/cm2) to bleach the activated fluorophores effectively. We then recorded individual fluorophores at different frame acquisition rates and measured their SNR (Fig. 3A). We found that the mean SNR was highest at 25 ms and 50 ms of acquisition (8.3 and 7.9, respectively). We therefore performed our live experiments consistently at 25 ms exposure time.


Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe.

Izeddin I, Specht CG, Lelek M, Darzacq X, Triller A, Zimmer C, Dahan M - PLoS ONE (2011)

Super-resolution dynamics: spatial versus temporal resolution.(A) Optimization of the SNR as a function of the image acquisition time. The left panels show the histograms of the amplitudes of the recorded signals at different acquisition times (12.5 ms to 500 ms); the right panels show their respective SNRs. The mean values are indicated as red lines. (B) PALM reconstruction of fixed dendritic spines from 500, 1000, 2000, and 5000 frames of 25 ms. (C) Standard deviation of the distribution of signals across a thin filopodium from the live recording shown in Fig. S2, as a function of the time window used for image reconstruction. (D) Time course of the number of detected molecules for ABP-tdEosFP and actin-PAmCherry at constant illumination. The arrow indicates an increase of the photoconversion laser power at the end of the experiment (5× for ABP-tdEosFP, 10× for actin-PAmCherry).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3022016&req=5

pone-0015611-g003: Super-resolution dynamics: spatial versus temporal resolution.(A) Optimization of the SNR as a function of the image acquisition time. The left panels show the histograms of the amplitudes of the recorded signals at different acquisition times (12.5 ms to 500 ms); the right panels show their respective SNRs. The mean values are indicated as red lines. (B) PALM reconstruction of fixed dendritic spines from 500, 1000, 2000, and 5000 frames of 25 ms. (C) Standard deviation of the distribution of signals across a thin filopodium from the live recording shown in Fig. S2, as a function of the time window used for image reconstruction. (D) Time course of the number of detected molecules for ABP-tdEosFP and actin-PAmCherry at constant illumination. The arrow indicates an increase of the photoconversion laser power at the end of the experiment (5× for ABP-tdEosFP, 10× for actin-PAmCherry).
Mentions: In order to determine the best conditions for live PALM imaging, we adjusted the image acquisition time to optimize the signal-to-noise-ratio (SNR) of the individual fluorophores. To ensure the fastest possible sampling of the structures, we chose the maximal excitation laser power (561 nm at 4 kW/cm2) to bleach the activated fluorophores effectively. We then recorded individual fluorophores at different frame acquisition rates and measured their SNR (Fig. 3A). We found that the mean SNR was highest at 25 ms and 50 ms of acquisition (8.3 and 7.9, respectively). We therefore performed our live experiments consistently at 25 ms exposure time.

Bottom Line: Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute.Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes.By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Kastler Brossel, CNRS UMR 8552, Department of Physics, École Normale Supérieure, Université Pierre et Marie Curie-Paris 6, Paris, France.

ABSTRACT
The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.

Show MeSH
Related in: MedlinePlus