![]() ![]() Although this position error has important implications for 2D SMACM techniques, the implications for 3D SMACM techniques are even more significant. Furthermore, fluorophores can be purposely anchored to convey orientation information about biological macromolecules, such as work in various SM studies on motor protein translocation ( 12– 14). However, in some cases, labels of biological structures can exhibit well-defined orientations ( 11). ![]() If labels are sufficiently rotationally mobile such that they explore much of the orientation space within a single acquisition, this effect is averaged away, and accuracy can be recovered. ( 10) noted that, with modest defocusing ( z = ☓00 nm), the position error associated with fitting to a centroid can exceed 100 nm for certain SM dipole orientations. Even more strikingly, the work by Engelhardt et al. ( 9) has shown that fitting such an SM image to a 2D Gaussian can result in position errors of tens of nanometers for molecules located in the microscope’s focal plane. However, immobile fluorescing SMs produce an inherently anisotropic emission pattern that depends on the orientation of the SM emission dipole moment relative to the optical axis ( 7, 8). Examples of these estimators include centroid finding, least-squares fitting to a 2D Gaussian function, and maximum likelihood methods that assume isotropic emitters. Typically, the SM fitting uses estimators that assume isotropic emission, i.e., that the center of the photon distribution of an SM image corresponds directly to the true position of the molecule. Collectively, these SM-based superresolution techniques can be grouped under the name SM Active Control Microscopy (SMACM), because they all rely on using various experimental strategies (photoactivation, switching, blinking additives, etc.) to maintain a very low concentration of emitters in each imaging frame, enabling the localization of SMs without overlap. Some of these techniques rely on precise localization of sparse subsets of single-molecule (SM) emitters to surpass the diffraction limit by up to an order of magnitude (precisions of tens of nanometers). The recent emergence of superresolution far-field optical microscopy techniques has provided a means for attaining resolution beyond the diffraction limit (∼250 nm) in noninvasive fluorescence imaging of biological structures ( 1, 2). Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (∼4× worse than the photon-limited precision 28 nm) to 34 nm (within 6 nm of the photon limit). 25 nm) to within 5 nm of photon-limited precision. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ∼2× worse than photon-limited precision (48 vs. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Failure to account for this fact can lead to significant lateral ( x, y) mislocalizations (up to ∼50–200 nm). However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule’s 3D orientation and z position. ![]() These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. ![]()
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