One of the main challenge in SMLM is to improve the axial positioning accuracy which is typically between 20 and 50 nm, and which gives only a relative positioning of the fluorophores. By coupling a dSTORM microscope with a detection module providing access to the supercritical angle fluorescence emission (SAF), the axial position of the fluorophore can be retrieved and gives the absolute distance regarding the glass slide supporting the cell. Only the detection path of the microscope is modify to insert a home made dual view module. The DONALD microscopy (Direct Optical Nanoscopy with Axially Localized Detection) achieves an isotropic resolution below 20 nm. This technique has just been the object of a publication in Nature Photonics http://www.nature.com/nphoton/journ…. A video summary is also available (https://youtu.be/Wf8iHYww9lY) or (https://vimeo.com/135072198).
The localization obtained with DONALD allows us to image the microtubules network labeled with Alexa 647 and in particular to evidence the hollowness.
3D image actin network labeled with the Alexa 488, on the left representing the diffraction limited image where the subnetwork is not discernable in contrast to the super-resolved DONALD image (right part of the image )
This work is developped in collaboration with Institut Langevin-ESPCI PArisTech (team E. Fort) et le Centre de photonique biomédicale de Paris Sud (G. Dupuis, S.Lécart). We were funded by the ANR and the DIM NanoK.