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The brain is characterized by an incredible molecular diversity, that underlies the complex structural specializations of neuronal cells as well as the functional intricacy of neuronal connectivity and synaptic transmission. Knowledge of the localization and positional relationship of the multitude of proteins orchestrating this diversity is essential to understand their roles. However, the fine processes of neurons contain a dense organization of components, and the diffraction limit of conventional optical microscopy (∼200 nm) precludes analyses of such information.
Super-resolution imaging methods overcome the diffraction limit and provide optical images of neuronal structures with unprecedented resolutions. Since its first application to neuroscience 14 years ago, super resolution microscopy has been used to dissect the molecular organization of active zones [1,2]; actin organization and membrane trafficking in growth cones [3,4] and dendritic spines protrusion [5]; synaptic transmission and plasticity at the single-molecule level [6]; the nanoscale arrangement of brain circuits [7]; the striking periodicity of the actin and spectrin cytoskeleton under the plasma membrane of axons [8,9,10].
Among all the super resolution imaging techniques, Single Molecule Localization Microscopy (SMLM) achieves the highest spatial resolution and is ideal for investigating individual molecules’ localization, interaction and dynamics, allowing the visualization of macro-molecular complexes in situ.
Abbelight implements single-molecule localization strategies (STORM, PALM, PAINT) in a powerful and simple-to-use imaging platform, providing neuroscientists with the tools to better understand the nanoscale molecular principles governing neuronal cells structure and function.
Abbelight features the highest 3D resolution (15 nm) over the largest field of view (200 mm²) in single molecule imaging, allowing the dissection of the sub-diffraction spectrin rings in individual axons over hundreds of microns (A and B, 3D view). In C, simultaneous multicolor imaging via spectral demixing easily separates with nanometric resolution the rings of actin-capping protein adducin (magenta) and β2 spectrin (yellow) alternating under the axonal membrane with a periodicity of ~190 nm.
The fine structure of the actin cytoskeleton in dendritic spines (D) is imaged using Abbelight ultra-fast technology; this permits to acquire statistically significant data on synaptic plasticity with the highest resolution and in the shortest times
Inhibitory (E) and excitatory synapses (D) are imaged using Abbelight multicolor technology; this permits to dissect with nanometer-level spatial resolution the pre- and post-synaptic structures. In E, GABAergic synapses in cultured cortical neurons are stained for postsynaptic Gephyrin and presynaptic RIM, imaged simultaneously. In F, the SNARE protein Vamp2 (red) and Glutamate transporters VGluT (green) of hippocampal varicose synapses are localized in mouse brain slices.
A through D: mouse hippocampal neurons were cultured for 14-21 days, fixed and stained as described in7. Adducin and b2-spectrin were immuno-stained with primary polyclonal antibodies and secondary antibodies conjugated with AF647 and CF680 fluorophores, compatible with multicolor STORM imaging with spectral demixing. Dendritic actin was stained with Phalloidin-AF647+.
E: mouse cortical neurons were cultured for 14-21 days, fixed and stained for simultaneous multicolor STORM imaging with spectral demixing : Gephyrin (green), was labelled with A647-conjugated antibodies and corresponds to the postsynaptic scaffold at inhibitory synapses; RIM1/2 (red) was labelled with CF680-conjugated antibodies and identifies the presynaptic active zone.
F: coronal cryosections of mouse Hippocampus were stained with VAMP2 ang VGluT primary antibodies, followed by secondary antibodies conjugated with AF647 and AF555 fluorophores, for sequential multicolor STORM.
3D imaging was performed on an Olympus Ix83 microscope with a 100X 1.49NA objective, equipped with Abbelight SAFe360 that implements enhanced astigmatism and DAISY technology for 3D isotropic super-resolution, and ASTER technology for large datasets. For STORM imaging coverslips were incubated in Abbelight SMART-Kit buffer.
Two-color STORM imaging was either performed by successive imaging with 647- and 532-nm lasers (AF647 and AF555), or simultaneously with 647-nm laser (AF647 and CF680). For the reconstructions, 30000-60000 frames were collected at 20 FPS (200×200 mm datasets), 100 FPS (50×50 mm datasets), or 300 FPS (35×35 mm datasets).
Single molecule localization, image reconstruction and spectral demixing of simultaneous multicolor datasets were performed in real time with Abbelight NEO software. NEO software was also used for 3D visualization and analysis of single molecule data, that is the spatial coordinates of each detected molecule.
Tools such as cluster analysis and single particle tracking are especially suited for nanoscale co-localization studies in crowded compartments like the active zone, and to analyze the dynamics of vesicular transport in axons and dendrites.
References
1. Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution Imaging of Chemical Synapses in the Brain. Neuron 68, 843–856 (2010).
2. Ehmann, N., Sauer, M. & Kittel, R. J. Super-resolution microscopy of the synaptic active zone. Front. Cell. Neurosci. 9, 7 (2015).
3. Nozumi, M., Nakatsu, F., Katoh, K. & Igarashi, M. Coordinated Movement of Vesicles and Actin Bundles during Nerve Growth Revealed by Superresolution Microscopy. Cell Rep. 18, 2203–2216 (2017).
4. Inavalli, V. V. G. K. et al. A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nat. Methods 16, 1263–1268 (2019)
5. Chazeau, A. et al. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion. EMBO J. 33, 2745–2764 (2014).
6. Pennacchietti, F. et al. Nanoscale molecular reorganization of the inhibitory postsynaptic density is a determinant of gabaergic synaptic potentiation. J. Neurosci. 37, 1747–1756 (2017).
7. Dudok, B. et al. Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling. Nat. Neurosci. 18, 75–86 (2015).
8. Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin and associated proteins form a periodic cytoskeletal structure in axons. Science (80-. ). (2013). doi:10.1021/nl061786n.Core-Shell
9. Leterrier, C. et al. Nanoscale Architecture of the Axon Initial Segment Reveals an Organized and Robust Scaffold. Cell Rep. 13, 2781–2793 (2015).
10. Vassilopoulos, S., Gibaud, S., Jimenez, A., Caillol, G. & Leterrier, C. Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings. Nat. Commun. 10, (2019).
Samples and images provided by:
Image A, B, C ;
Samples and images courtesy of Christophe Leterrier and Karoline Friedl, NeurocytoLab, INP, Univ. Aix-Marseille/CNRS
Image D
Sample and image courtesy of Christophe Leterrier, Karoline Friedl and Florian Wernert, NeurocytoLab, INP, Univ. Aix-Marseille/CNRS,
Image E,F
Samples courtesy of Christian Specht, ENS/INSERM (E panel) and Veronique Bernard, IBPS, UPMC/INSERM Paris (F)