Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function.
We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.
Pavani
Sri Rama Prasanna
SR
Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA.
Thompson
Michael A
MA
Biteen
Julie S
JS
Lord
Samuel J
SJ
Liu
Na
N
Twieg
Robert J
RJ
Piestun
Rafael
R
Moerner
W E
WE
D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*