Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics”

Li et al. (Research Articles, 28 August 2015, aab3500) purport to present solutions to long-standing challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods’ reliability to visualize specimen features at sub–100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.

[1]  S. Hell.  Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[2]  A. Stemmer,et al.  True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Christian Eggeling,et al.  Nanoscopy of Living Brain Slices with Low Light Levels , 2012, Neuron.

[4]  Christian Eggeling,et al.  Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Hell,et al.  Imaging and writing at the nanoscale with focused visible light through saturable optical transitions , 2003 .

[6]  J. J. Macklin,et al.  Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution , 2011, Proceedings of the National Academy of Sciences.

[7]  Christian Eggeling,et al.  Diffraction-unlimited all-optical imaging and writing with a photochromic GFP , 2011, Nature.

[8]  Stefan W. Hell,et al.  Far-field fluorescence nanoscopy of diamond color centers by ground state depletion , 2009 .

[9]  Christian Eggeling,et al.  A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching , 2011, Nature Biotechnology.

[10]  M. Gustafsson.  Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Stefan W Hell,et al.  STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. , 2015, Cell reports.

[12]  M. Davidson,et al.  Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics , 2015, Science.

[13]  S. Hell,et al.  Wide‐field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching , 2007, Microscopy research and technique.

[14]  Peter Dedecker,et al.  Diffraction-unlimited imaging: from pretty pictures to hard numbers , 2015, Cell and Tissue Research.

[15]  M. Gustafsson.  Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy , 2000, Journal of microscopy.

[16]  Marcus Dyba,et al.  Concepts for nanoscale resolution in fluorescence microscopy , 2004, Current Opinion in Neurobiology.

[17]  R. Heintzmann,et al.  Saturated patterned excitation microscopy--a concept for optical resolution improvement. , 2002, Journal of the Optical Society of America. A, Optics, image science, and vision.