Two-color far-field fluorescence nanoscopy.
We demonstrate two-color fluorescence microscopy with nanoscale spatial resolution by applying stimulated emission depletion on fluorophores differing in their absorption and emission spectra. Green- and red-emitting fluorophores are selectively excited and quenched using dedicated beam pairs. The stimulated emission depletion beams deliver a lateral resolution of <30 nm and 65 nm for the green and the red color channel, respectively. The approximately 5 nm alignment accuracy of the two images establishes the precision with which differently labeled proteins are correlated in space. Colocalized nanoscopy is demonstrated with endosomal protein patterns and by resolving nanoclusters of a mitochondrial outer membrane protein, Tom20, in relation with the F(1)F(0)ATP synthase. The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells.
Donnert
Gerald
G
Keller
Jan
J
Wurm
Christian A
CA
Rizzoli
Silvio O
SO
Westphal
Volker
V
Schönle
Andreas
A
Jahn
Reinhard
R
Jakobs
Stefan
S
Eggeling
Christian
C
Hell
Stefan W
SW
D000818:Animals; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D007089:Image Enhancement / Q000379:methods*; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism* / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods*; D016716:PC12 Cells; D006180:Proton-Translocating ATPases / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism* / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C489998:Tomm20 protein, rat; D006180:Proton-Translocating ATPases