Mikael P. Backlund

Simultaneous, accurate measurement of the 3D position and orientation of single molecules

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 photon-limited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

Rotational Mobility of Single Molecules Affects Localization Accuracy in Super-Resolution Fluorescence Microscopy

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.

Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions.

We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

The Role of Molecular Dipole Orientation in Single‐Molecule Fluorescence Microscopy and Implications for Super‐Resolution Imaging

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.

Removing orientation-induced localization biases in single-molecule microscopy using a broadband metasurface mask

Nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques, including single-molecule tracking and wide-field super-resolution imaging 1. To date, a central consideration of such techniques is how to optimize the precision of molecular localization. However, as these methods continue to push toward the nanometre size scale, an increasingly important concern is the localization accuracy. In particular, single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole, which can cause significant localization biases using common estimators 2-5. Here we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the Fourier plane of the microscope. We do so using a high efficiency dielectric metasurface polarization/phase device composed of nanoposts with sub-wavelength spacing 6. The method is demonstrated both on fluorophores embedded in a polymer matrix, and in dL5 protein complexes that bind Malachite green 7, 8.

Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope

The double-helix point spread function microscope is used to track single pairs of fluorescently labeled chromosomal loci in live yeast cells in three dimensions. Enhanced velocity cross-correlations are observed between pairs of GAL loci in diploid cells under repressive conditions, and ubiquitous subdiffusive exponents are found to be near 0.6–0.75.

Cytoplasmic RNA-Protein Particles Exhibit Non-Gaussian Subdiffusive Behavior

The cellular cytoplasm is a complex, heterogeneous environment (both spatially and temporally) that exhibits viscoelastic behavior. To further develop our quantitative insight into cellular transport, we analyze data sets of mRNA molecules fluorescently labeled with MS2-GFP tracked in real time in live Escherichia coli and Saccharomyces cerevisiae cells. As shown previously, these RNA-protein particles exhibit subdiffusive behavior that is viscoelastic in its origin. Examining the ensemble of particle displacements reveals a Laplace distribution at all observed timescales rather than the Gaussian distribution predicted by the central limit theorem. This ensemble non-Gaussian behavior is caused by a combination of an exponential distribution in the time-averaged diffusivities and non-Gaussian behavior of individual trajectories. We show that the non-Gaussian behavior is a consequence of significant heterogeneity between trajectories and dynamic heterogeneity along single trajectories. Informed by theory and simulation, our work provides an in-depth analysis of the complex diffusive behavior of RNA-protein particles in live cells.

The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation

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).

Motion of chromosomal loci and the mean-squared displacement of a fractional Brownian motion in the presence of static and dynamic errors

Mean-squared displacement (MSD) analysis is one of the most prevalent tools employed in the application of single-particle tracking to biological systems. In camera-based tracking, the effects of “static error” due to photon fluctuations and “dynamic error” due to motion blur on the MSD have been well-characterized for the case of pure Brownian motion, producing a known constant offset to the straight-line MSD. However, particles tracked in cellular environments often do not undergo pure Brownian motion, but instead can for instance exhibit anomalous diffusion wherein the MSD curve obeys a power law with respect to time, MSD=2D*τα, where D* is an effective diffusion coefficient and 0 < α ≤ 1. There are a number of models that can explain anomalous diffusive behavior in different subcellular contexts. Of these models, fractional Brownian motion (FBM) has been shown to accurately describe the motion of labeled particles such as mRNA and chromosomal loci as they traverse the cytoplasm or nucleoplasm (i.e. crowded viscoelastic environments). Despite the importance of FBM in biological tracking, there has yet to be a complete treatment of the MSD in the presence of static and dynamic errors analogous to the special case of pure Brownian motion. We here present a closed-form, analytical expression of the FBM MSD in the presence of both types of error. We have previously demonstrated its value in live-cell data by applying it to the study of chromosomal locus motion in budding yeast cells. Here we focus on validations in simulated data.

The Double-Helix Microscope Enables Precise and Accurate Measurement of 3D Single-Molecule Orientation and Localization Beyond the Diffraction Limit

Failure to account for the asymmetric nature of fluorophore emission in single-molecule super-resolution microscopy can lead to large localization errors. We use the Double-Helix microscope to correct such mislocalizations while simultaneously extracting 3D position and molecular orientation.