Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. 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. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ∼50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. 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. 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. 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. 25 nm) to within 5 nm of photon-limited precision. 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 photonlimited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). We describe in detail how the localization precision quantitatively depends upon the number of photons detected and the z position of the nanoscale emitter, thereby showing a ~10 nm localization capability along x, y, and z in the limit of weak emitters. Experimental measurements are compared to Fisher information calculations of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions.A critical problem in nanoscale science is the need to noninvasively and from a distance determine the location of a nanoscale object in three dimensions. Even though the diffraction limit of visible light is on the order of 200 nm, the two-dimensional (2D) image of a single molecule can be fit to find the x-y position of the molecule with nanometer precision1 , 2. The localization precision scales roughly as σ/(N) 1/2 where σ is the diffraction-limited standard deviation of the microscope point spread function (PSF) and N is the number of photons detected from a point-like emitter. This "super-localization" property, well-known in many areas of science 3 , was used to great effect by many groups to track isolated point-like singlemolecule emitters in a variety of contexts 4 , 5. However, imaging a continuous structure requires a high density of fluorescent labels, and the nanoscale detail is lost because the PSFs overlap. By using photoswitchable or photoactivatable fluorophores to keep the concentration of active emitters low at any one time, super-resolution images can be extracted by successive single-molecule localizations 6 -8 . These methods were, until recently, mostly limited to 2D imaging. Unfortunately, three-dimensional (3D) position information is difficult to obtain using a diffraction-limited conventional fluorescence microscope for two reasons: (1) the PSF is symmetric about the focal plane meaning that a molecule × nm above the focal plane cannot be distinguished from a molecule × nm below the focal plane, and (2) the PSF contains little information about the axial position of the emitter for a few hundred nanometers about the focal plane 9, 10, meaning that it is quite difficult to super-localize molecules that are in the focal plane of the microscope. Recently, several solutions to these problems have been described including using astigmatism 11 -13, imaging in two different focal planes 14,10,15,16 and interferometry 17, 18 . Fast 3D fluorescent particle tracking has also been performed using confocal imaging [19][20][21] and by using two rotating laser beams to track single quantum dots 21 . We recently demonstrated that a Double-Helix Point Spread Function (DH-PSF) can be used to super-localize single molecules in three dimensions 22 . Combining...

Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, livecell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 × 12 μm).cell biology | wide-field microscopy | cytoskeleton | fluorescence microscopy | bacteria

Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.

The asymmetric nature of single-molecule (SM) dipole emission patterns limits the accuracy of position determination in localization-based super-resolution fluorescence microscopy. The degree of mislocalization depends highly on the rotational mobility of SMs; only for SMs rotating within a cone half angle α > 60° can mislocalization errors be bounded to ≤ 10 nm. Simulations demonstrate how low or high rotational mobility can cause resolution degradation or distortion in super-resolution reconstructions.

Wide-field microscopy with a double-helix point spread function (DH-PSF) provides three-dimensional (3D) position information beyond the optical diffraction limit. We compare the theoretical localization precision for an unbiased estimator of the DH-PSF to that for 3D localization by astigmatic and biplane imaging using Fisher information analysis including pixelation and varying levels of background. The DH-PSF results in almost constant localization precision in all three dimensions for a 2 μm thick depth of field while astigmatism and biplane improve the axial localization precision over smaller axial ranges. For high signal-to-background ratio, the DH-PSF on average achieves better localization precision.

Optical coherence tomography (OCT) is a powerful biomedical imaging technology that relies on the coherent detection of backscattered light to image tissue morphology in vivo. As a consequence, OCT is susceptible to coherent noise (speckle noise), which imposes significant limitations on its diagnostic capabilities. Here we show speckle-modulating OCT (SM-OCT), a method based purely on light manipulation that virtually eliminates speckle noise originating from a sample. SM-OCT accomplishes this by creating and averaging an unlimited number of scans with uncorrelated speckle patterns without compromising spatial resolution. Using SM-OCT, we reveal small structures in the tissues of living animals, such as the inner stromal structure of a live mouse cornea, the fine structures inside the mouse pinna, and sweat ducts and Meissner’s corpuscle in the human fingertip skin—features that are otherwise obscured by speckle noise when using conventional OCT or OCT with current state of the art speckle reduction methods.

Fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting single-molecule localization microscopy (SMLM) to measure their orientations and rotational mobilities with high precision remains a challenge. Inspired by dipole radiation patterns, we design and implement a Tri-spot point spread function (PSF) that simultaneously measures the three-dimensional orientation and the rotational mobility of dipole-like emitters across a large field of view. We show that the orientation measurements done using the Tri-spot PSF are sufficiently accurate to correct the anisotropy-based localization bias, from 30 nm to 7 nm, in SMLM. We further characterize the emission anisotropy of fluorescent beads, revealing that both 20-nm and 100-nm diameter beads emit light significantly differently from isotropic point sources. Exciting 100-nm beads with linearly polarized light, we observe significant depolarization of the emitted fluorescence using the Tri-spot PSF that is difficult to detect using other methods. Finally, we demonstrate that the Tri-spot PSF detects rotational dynamics of single molecules within a polymer thin film that are not observable by conventional SMLM.