Near-isotropic 3D optical nanoscopy with photon-limited chromophores.
Imaging approaches based on single molecule localization break the diffraction barrier of conventional fluorescence microscopy, allowing for bioimaging with nanometer resolution. It remains a challenge, however, to precisely localize photon-limited single molecules in 3D. We have developed a new localization-based imaging technique achieving almost isotropic subdiffraction resolution in 3D. A tilted mirror is used to generate a side view in addition to the front view of activated single emitters, allowing their 3D localization to be precisely determined for superresolution imaging. Because both front and side views are in focus, this method is able to efficiently collect emitted photons. The technique is simple to implement on a commercial fluorescence microscope, and especially suitable for biological samples with photon-limited chromophores such as endogenously expressed photoactivatable fluorescent proteins. Moreover, this method is relatively resistant to optical aberration, as it requires only centroid determination for localization analysis. Here we demonstrate the application of this method to 3D imaging of bacterial protein distribution and neuron dendritic morphology with subdiffraction resolution.
Tang
Jianyong
J
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA. tangjy@gmail.com
Akerboom
Jasper
J
Vaziri
Alipasha
A
Looger
Loren L
LL
Shank
Charles V
CV
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D036103:Nanotechnology; D055096:Optical Devices; D017785:Photons; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins
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