Dual-color 3D PALM/dSTORM imaging of centrosomal proteins using MicAO 3DSR

Grégory Clouvel; Audrius Jasaitis; James Sillibourne; Ignacio Izeddin; Mohamed El Beheiry; Xavier Levecq; Maxime Dahan; Michel Bornens; Xavier Darzacq

doi:10.1117/12.2001986

22 February 2013 Dual-color 3D PALM/dSTORM imaging of centrosomal proteins using MicAO 3DSR

Grégory Clouvel, Audrius Jasaitis, James Sillibourne, Ignacio Izeddin, Mohamed El Beheiry, Xavier Levecq, Maxime Dahan, Michel Bornens, Xavier Darzacq

Author Affiliations +

Proceedings Volume 8590, Single Molecule Spectroscopy and Superresolution Imaging VI; 85900Z (2013) https://doi.org/10.1117/12.2001986
Event: SPIE BiOS, 2013, San Francisco, California, United States

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.

Citation Download Citation

Grégory Clouvel, Audrius Jasaitis, James Sillibourne, Ignacio Izeddin, Mohamed El Beheiry, Xavier Levecq, Maxime Dahan, Michel Bornens, and 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); https://doi.org/10.1117/12.2001986

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available