Mapping molecules in scanning far-field fluorescence nanoscopy.
In fluorescence microscopy, the distribution of the emitting molecule number in space is usually obtained by dividing the measured fluorescence by that of a single emitter. However, the brightness of individual emitters may vary strongly in the sample or be inaccessible. Moreover, with increasing (super-) resolution, fewer molecules are found per pixel, making this approach unreliable. Here we map the distribution of molecules by exploiting the fact that a single molecule emits only a single photon at a time. Thus, by analysing the simultaneous arrival of multiple photons during confocal imaging, we can establish the number and local brightness of typically up to 20 molecules per confocal (diffraction sized) recording volume. Subsequent recording by stimulated emission depletion microscopy provides the distribution of the number of molecules with subdiffraction resolution. The method is applied to mapping the three-dimensional nanoscale organization of internalized transferrin receptors on human HEK293 cells.
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany.
Haisen
H
Ta
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany.
Jan
0000000327530848
J
Keller
1] Statistical Inverse Problems in Biophysics Group, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany [2] Department of Mathematics, University of Innsbruck, Innsbruck 6020, Austria.
Markus
M
Haltmeier
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen 37073, Germany.
Sinem K
SK
Saka
NanoBioSciences Group, Institute of Physical and Theoretical Chemistry, Braunschweig University of Technology, Braunschweig 38106, Germany.
Jürgen
J
Schmied
Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen 37073, Germany.
Felipe
F
Opazo
NanoBioSciences Group, Institute of Physical and Theoretical Chemistry, Braunschweig University of Technology, Braunschweig 38106, Germany.
Philip
P
Tinnefeld
1] Statistical Inverse Problems in Biophysics Group, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany [2] Institute for Mathematical Stochastics and Felix Bernstein Institute for Mathematical Statistics in the Biosciences, University of Göttingen, Göttingen 37077, Germany.
Axel
A
Munk
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany.
Stefan W
SW
Hell
D052157:Aptamers, Nucleotide; D004247:DNA / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D061925:Immobilized Nucleic Acids / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D013194:Staining and Labeling
D052157:Aptamers, Nucleotide; D061925:Immobilized Nucleic Acids; D004247:DNA