22 February 2013 Dual-color 3D PALM/dSTORM imaging of centrosomal proteins using MicAO 3DSR
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Abstract
The determination of 3-dimensional arrangement of subcellular assemblies has become a necessary requirement in cellular biology. Unfortunately, the size of most assemblies lies beyond the diffraction limit and therefore they cannot be visualized using conventional fluorescence microscopy techniques. Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) enable the localization of fluorescent molecules with nanometric resolution. We used these microscopy methods together with MicAO 3D-SR – the first adaptive optics device which introduces the three-dimensional imaging capability for PALM/STORM. MicAO also corrects various types of aberrations induced by optical elements inside the microscope and by the biological sample. The correction of these aberrations almost doubles the number of detected photons, increasing the localization precision of PALM/STORM by 40%. At 1000 detected photons the localization precision of our setup is 8 nm in lateral and 16 nm in axial directions. The separate optimization performed for two different colors delivers superb imaging quality, as demonstrated by dual-color 3-dimentional imaging of two centrosomal proteins in HeLa cells.
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Grégory Clouvel, Audrius Jasaitis, James Sillibourne, Ignacio Izeddin, Mohamed El Beheiry, Xavier Levecq, Maxime Dahan, Michel Bornens, Xavier Darzacq, "Dual-color 3D PALM/dSTORM imaging of centrosomal proteins using MicAO 3DSR", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900Z (22 February 2013); doi: 10.1117/12.2001986; https://doi.org/10.1117/12.2001986
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