Oumeng Zhang Nano Letters
Oumeng Zhang Nano Letters – New results Solution of 3D rotational and translational dynamics of individual molecules using radially and azimuthally polarized fluorescence
View ORCID Profile Oumeng Zhang , View ORCID Profile Weiyan Zhou , View ORCID Profile Jin Lu , View ORCID Profile Tingting Wu , View ORCID Profile Matthew D. Lew
Oumeng Zhang Nano Letters
† Department of Electrical and Systems Engineering ‡ Center for Living Systems Science and Engineering ¶ Institute of Materials Science and Engineering, Washington University in St. Louis, Missouri 63130, USA
Single Molecules Are Your Quanta: A Bottom Up Approach Toward Multidimensional Super Resolution Microscopy
We describe a radially and azimuthally polarized (raPol) microscope for high-throughput detection and estimation in single-molecule orientation-localization microscopy (SMOLM). With 5,000 photons detected from Nile Red (NR) transiently bound in supported lipid bilayers (SLBs), raPol SMOLM achieves localization accuracies of 2.1 nm, 1.4
Orientation accuracy and an accuracy of 0.15 sr when estimating rotational wobble. Within the DPPC SLB, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NRs from freely exploring all orientations. Treatment of SLB with methylβ-cyclodextrin-loaded cholesterol (MpCD-chol) causes the orientational diffusion of NRs to be dramatically reduced, but surprisingly, the mean lateral displacements of NRs drastically increase from 20.8 nm to 75.5 nm (200 ms time delay). These jump diffusion events overwhelmingly originate from cholesterol-rich nanodomains in SLBs. These detailed single-molecule rotational and translational dynamics measurements are enabled by the high precision of raPol measurements and are not detectable in standard SMLM.
Simultaneous imaging of the positions and orientations of molecules in single-molecule orientational localization microscopy (SMOLM) provides unparalleled insight into biochemical processes. Recent developments in SMOLM allow scientists to resolve the organization of amyloid aggregates,
However, many SMOLM techniques exhibit low measurement accuracy for molecules that are oriented out of the plane of the coverslip (e.g.
Six Dimensional Single Molecule Imaging With Isotropic Resolution Using A Multi View Reflector Microscope
). Others improve measurement accuracy by significantly expanding the PSF imaging system and thus suffer from degraded localization accuracy and detection sensitivity for weak emitters (e.g.
For imaging the 3D positions and orientations of single molecules (SM), no existing techniques show sufficiently high localization accuracy, orientation accuracy, and sensitivity to SM fluctuations for simultaneous 3D orientation, fluctuation, and tracking of SM position in lipid membranes. Inspired by the symmetry in the dipole emission diagram, we demonstrate a highly sensitive and easy-to-implement SMOLM technique using radially and azimuthally polarized (raPol) fluorescence,
It is implemented using a commercially available vortex (half-wave) plate (VWP) and polarizing beam splitter (PBS) within a wide-field epifluorescence microscope. Unlike other techniques, the orientation estimation performance of the raPol microscope is excellent for molecules oriented along the optical axis (out of the plane of the coverslip). We use raPol PSF to study the dynamics of Nile red (NR) molecules transiently bound to supported lipid bilayers (SLBs).
Imaging the dynamics of NRs in DPPC SLBs reveals the existence of randomly oriented binding pockets that prevent the free rotation of NRs. In addition, raPol is able to simultaneously monitor the position and orientation of NRs when probing SLBs modified with cholesterol-loaded methyl-β-cyclodextrin (MβCD-chol). As cholesterol (chol) is deposited, the orientation of the NR tilts mostly perpendicular to the SLB and its rotational diffusion is greatly reduced, but its translational diffusion dramatically increases almost fourfold. These data suggest that NR “jumps” between cholesterol-rich nanodomains within the SLB. To our knowledge, these experiments are the first measurements of how MβCD-chol affects the nanoscale chemical environments in lipid membranes at the single-molecule level.
Simultaneous Orientation And 3d Localization Microscopy With A Vortex Point Spread Function
Represents a freely swinging molecule. Here we assume that the SM oscillates uniformly in the cone for simplicity, and our analysis can be easily adapted for other rotating potential wells or geometries.
Radially and azimuthally polarized (raPol) standard PSF. (a) The rotational diffusion of a fluorescent molecule is described by a unit vector µ and a solid ripple angle Ω on the surface of a hard-edged cone. (b) A vortex (half) wave plate (VWP) is located at the back focal plane of the imaging system, transforming radially and azimuthally polarized light into
Polarized light, or The arrows represent the VWP fast axis. (c) Representative radially (red) and azimuthally (blue) polarized PSFs of spin-fixed molecules with polar angles
90°, 45° and 0°. Colorbar: normalized intensity. Scale: 500 nm. (d) Probability of failed detection
Single Molecule Localization Microscopy Of 3d Orientation And Anisotropic Wobble Using A Polarized Vortex Point Spread Function
(i.e., false negative) for 250 signal photons detected from SM with a fixed fluctuation of Ω = 0.28
. These probabilities were calculated using a Monte Carlo simulation of 25,000 noisy images. Best possible accuracy for estimating (e) average orientation µ, (f) corrugation angle Ω and (g) 2D position r SM with 5000 signal photons detected based on Crámer–Rao coupling. Orange:
Due to the symmetry of the dipole emission pattern, the photons emitted by the SM are oriented parallel to the optical axis (
Therefore, an intuitive way to distinguish out-of-plane molecules from in-plane molecules is to measure radially vs. azimuthally polarized fluorescence. We achieve this polarization separation by adding a VWP (Figure 1b) to the back focal plane (BFP) of the microscope with two polarized imaging channels. The spatially varying direction of the VWP fast axis turns the radially and azimuthally polarized light to
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Polarized light. PBS is then used to separate the fluorescence into radially and azimuthally polarized images (Figure S1a). When a molecule rotates out of plane, i.e
The photons shift from the azimuthally polarized to the radially polarized channel. When the SM is aligned with the optical axis, all fluorescence photons are confined to a radially polarized image (Figure 1c, red).
A high detection rate is critical for collecting unbiased measurements of SM dynamics. We quantify the false detection (false negative) rate.
(SI Section 2.1). Note that due to the complexity of joint detection and localization of molecules in SMLM and SMOLM, the theoretical rate
Super Resolution Microscopy With Single Molecules In Biology And Beyond–essentials, Current Trends, And Future Challenges
Is overly optimistic compared to the practical performance of the algorithm and should be interpreted as an estimate of the relative performance between PSFs. For low signal-to-background ratio (SBR), we found that the detection performance of raPol is comparable to
Half (Figure 1d). Thus, weak SMs are detected more reliably with raPol in contrast to most proposed SMOLM-optimized PSFs; it is more difficult to detect faint emitters with larger PSFs. Due to the toroidal, polarized emission pattern of the SM, the detection performance is orientation dependent. For
Pol because the energy is concentrated in the radially polarized channel, but worse for in-plane molecules for the opposite reason (Figure 1c). Intuitively, the missed detection rate decreases as the PSF peak intensity increases (Figure S2b,c). Interestingly, due to the symmetry of raPol, its detection performance is not affected by the azimuthal angle
The accuracy of the mean orientation measurement is quantified as the angle subtended by the arc, which represents the measurement uncertainty on the orientation unit sphere ( SI Section 2.2 ). Surprisingly, raPol exhibits excellent orientation measurement accuracy
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Pol for 91.0% of all possible SM orientations (Figure 1e and Figure S2e). Similarly, for 83.8% of all possible orientations, raPol SM measures wobble more accurately than
Pol does (Figure 1f and Figure S2f). Notably, the average 2D localization accuracy using raPol is also slightly better than u
While the translational dynamics of fluorophore-lipid interactions have been characterized at the SM level, e.g., by fluorescence correlation spectroscopy,
Relatively little is known about their rotational dynamics. We further study the rotational dynamics of Nile red in SLB
Polarization Spectroscopy Of Single Fluorescent Molecules
We form a bilayer of DPPC [di(16:0)PC (phosphatidylcholine), Figure S4] on a coverslip and use RoSE-O, a sparseness-promoting maximum likelihood estimation algorithm,
Analyze images of blinking NR molecules and extract their positions and orientations. First, we measure the NR within the DPPC SLB using both
Pol and raPol, excited by circularly polarized epi (Figure 2a) and total internal reflection fluorescence (TIRF, Figure 2d) illumination. Interestingly, the polar orientation of NR
Varies significantly with illumination polarization, from the median to , implying that the orientation of the emission dipole of NR correlates with that of its absorption dipole moment within the DPPC SLB. To confirm, we changed the slope of the laser beam to
Transforming Separation Science With Single Molecule Methods
Polarized electric field in the excitation beam increased, the observed NR polar angles systematically decreased. The change in polar angle is evident in the raPol images at every illumination angle
Rotational diffusion of Nile red (NR) within DPPC supported lipid bilayers (SLB). (a-d) (i) Averaged raPol images and (ii) measurements of orientation and NR fluctuations as a function of illumination tilt angle
: (a) 0°, (b) 25°, (c) 45° and (d) total internal reflection fluorescence (TIRF). Measurements were collected using both (orange)
Displaying Pol and (green) raPol. Crosses represent the mean of the distribution, while solid lines represent the 33rd and 67th percentiles, and dotted lines represent the 10th and 90th percentiles. (e, f) Quantification of the effect of illumination tilt angles
Polarization Encoded Colocalization Microscopy At Cryogenic Temperatures
And (e) Ω fluctuations. Green: experimental measurement. Dots represent the median, solid lines represent the 33rd and 67th percentiles, and dotted lines represent the 10th and 90th percentiles. Black and gray: simulated orientation measurements of rotationally diffusive SMs, assuming an excited-state lifetime of 4.6 ns, rotational diffusion coefficient of 0.035 rad
] sr. The lines, dark and light gray areas represent the median, 33/67. and 10/90 percentile. Colorbar: photons; scale bar: 500 nm.
To explain how the polarization of the illumination laser strongly affects the observed orientations of NRs, we used Monte Carlo simulations to model light absorption, rotational diffusion,
And photon emission of NR within the DPPC SLB, using reported values for its excited-state lifetime (4.6 ns)