Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction
limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the
position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or
2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the
molecule’s transition dipole moment, as well as its z-position. Failure to account for this fact can result in localization
errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range
becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically
employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and
accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF)
microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations
in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited
precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard
deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).
© (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Mikael P. Backlund ; Matthew D. Lew ; Adam S. Backer ; Steffen J. Sahl ; Ginni Grover, et al.
The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation
", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900L (February 22, 2013); doi:10.1117/12.2001671; http://dx.doi.org/10.1117/12.2001671