Imaging intracellular fluorescent proteins at nanometer resolution.
We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.
Betzig
Eric
E
Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147, USA. betzige@janelia.hhmi.org
Patterson
George H
GH
Sougrat
Rachid
R
Lindwasser
O Wolf
OW
Olenych
Scott
S
Bonifacino
Juan S
JS
Davidson
Michael W
MW
Lippincott-Schwartz
Jennifer
J
Hess
Harald F
HF
D000199:Actins / Q000032:analysis; D000465:Algorithms; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002522:Chlorocebus aethiops; D005453:Fluorescence; D022001:Focal Adhesions / Q000737:chemistry; D015683:Gene Products, gag / Q000032:analysis; D015497:HIV-1; D008027:Light; D008164:Luminescent Proteins / Q000032:analysis*; D008247:Lysosomes / Q000737:chemistry; D008853:Microscopy / Q000379:methods*; D008928:Mitochondria / Q000737:chemistry; D036103:Nanotechnology; D015388:Organelles / Q000737:chemistry*; D038761:Photobleaching; D011506:Proteins / Q000032:analysis*; D011554:Pseudopodia / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D016596:Vinculin / Q000032:analysis
D000199:Actins; D015683:Gene Products, gag; D008164:Luminescent Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016596:Vinculin