Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay

Sun, Brian; Ding, Tianben; Zhou, Weiyan; Porter, Tara S.; Lew, Matthew D.

doi:10.1021/acs.nanolett.4c01263

Cited by 2 publications

(1 citation statement)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Perhaps most importantly, the development of brighter fluorophores − will yield higher SBRs that are necessary for high-precision wobble measurements. We eagerly anticipate continued development of single-molecule imaging technologies to enable cutting-edge biophysical experiments. − …”

Section: Discussionmentioning

confidence: 99%

Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

Zhou,

Wu,

Lew

2024

J. Phys. Chem. A

Self Cite

View full text Add to dashboard Cite

Many biophysical techniques, such as singlemolecule fluorescence correlation spectroscopy, Forster resonance energy transfer, and fluorescence anisotropy, measure the translation and rotation of biomolecules to quantify molecular processes at the nanoscale. These methods often simplify data analysis by assuming isotropic rotational diffusion, e.g., that molecules wobble within a circular cone. This simplification ignores the anisotropy present in many biological contexts that may cause molecules to exhibit different degrees of diffusion in different directions. Here, we loosen this assumption and establish a theoretical framework for describing and measuring anisotropic rotational diffusion using fluorescence imaging. We show that anisotropic wobble is directly quantified by the eigenvalues of a 3-by-3 positive-semidefinite Hermitian matrix M consisting of the second-order moments of a molecule's transition dipole μ. This formalism enables us to model the influence of unavoidable shot noise using a Hermitian perturbation matrix E; the eigenvalues of E directly bound errors in measurements of wobble via Weyl's inequality. Quantifying various perturbations E reveals that anisotropic wobble measurements are generally more sensitive to errors compared to quantifying isotropic wobble. Moreover, severe shot noise can induce negative eigenvalues in estimates of M, thereby causing the anisotropic wobble measurement to fail. Our analysis, using Fisher information, shows that techniques with worse orientation measurement sensitivity experience stronger perturbations E and require larger signal to background ratios to measure anisotropic rotational diffusion accurately. Our work provides deep insights for improving the state of the art in imaging the orientations and anisotropic rotational diffusion of single molecules.

show abstract

Section: Discussionmentioning

confidence: 99%

Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

Zhou,

Wu,

Lew

2024

J. Phys. Chem. A

Self Cite

View full text Add to dashboard Cite

show abstract

Event-based Single Molecule Localization Microscopy (eventSMLM) for High Spatio-Temporal Super-resolution Imaging

Basumatary,

Aravinth,

Pant

et al. 2023

Preprint

View full text Add to dashboard Cite

Photon emission by single molecules is a random event that has a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing the arrival time of photons and their distribution (emission characteristics) within a single blinking period (typically, ~30 ms) of a single molecule. This information can be used to better localize single molecules within a user-defined collection time (shorter than average blinking time) of the event detector. The events collected over every short interval of time (~ 3 ms) give rise to several independent photon distributions (PSFs) of single molecules in the event camera. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period than the entire blinking period gives rise to several realizations of single-molecule PSFs. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule PSF give several position estimates of the single-molecules, leading to multiple PSF centroids. Fitting these centroid points by a circle gives an approximate position (circle center) and geometric localization precision (circle area) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the photon detection events and the recorded PSF, the estimated value is purely experimental rather than theoretical (Thomson's formula). Moreover, this eliminates the need for noise calculation and background estimation. The method is tested on three different test samples (1) Scattered Cy3 dye molecules on a coverslip, (2) Mitochondrial network in a cell, and (3) Dendra2HA transfected NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events / photons. Experimental results on transfected NIH3T3 cells show a localization precision of ~ 10 nm, which is ~ 3.5 fold better than standard SMLM. Results reveal a spatio-temporal resolution (lp × t) of 122.5p (where, p=10-12meter.second) (measured in terms of localization precision). Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. Moreover, single-molecule imaging on live cells reveals the temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over a period of 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy capable of high spatio-temporal super-resolution microscopy (in the range ~ 1p = 10-12meter.sec).

show abstract

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay

Cited by 2 publications

References 52 publications

Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

Event-based Single Molecule Localization Microscopy (eventSMLM) for High Spatio-Temporal Super-resolution Imaging

Contact Info

Product

Resources

About