POLCAM: Instant Molecular Orientation Microscopy for the Life Sciences

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2024 Oct 7

PMID 39375574

Authors

Ezra Bruggeman

Oumeng Zhang

Lisa-Maria Needham

Markus Korbel

Sam Daly

Matthew Cheetham

Ruby Peters

Tingting Wu

Andrey S Klymchenko

Simon J Davis

Ewa K Paluch

David Klenerman

Matthew D Lew

Kevin OHolleran

Steven F Lee

Affiliations

Soon will be listed here.

Abstract

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, which can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field-of-view errors, used simulations to optimize experimental design and developed a fast algorithm based on Stokes parameter estimation that can operate over 1,000-fold faster than the state of the art, enabling near-instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study α-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

Citing Articles

Single-molecule orientation-localization microscopy: Applications and approaches.

Zhang O, Lew M Q Rev Biophys. 2024; 57:e17.

PMID: 39710866 PMC: 11771422. DOI: 10.1017/S0033583524000167.

RASP: Optimal Single Puncta Detection in Complex Cellular Backgrounds.

Fu B, Brock E, Andrews R, Breiter J, Tian R, Toomey C J Phys Chem B. 2024; 128(15):3585-3597.

PMID: 38593280 PMC: 11033865. DOI: 10.1021/acs.jpcb.4c00174.

Supramolecular polymers form tactoids through liquid-liquid phase separation.

Fu H, Huang J, van der Tol J, Su L, Wang Y, Dey S Nature. 2024; 626(8001):1011-1018.

PMID: 38418913 PMC: 10901743. DOI: 10.1038/s41586-024-07034-7.

References

Betzig E, Patterson G, Sougrat R, Lindwasser O, Olenych S, Bonifacino J . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006; 313(5793):1642-5. DOI: 10.1126/science.1127344. View

Rust M, Bates M, Zhuang X . Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006; 3(10):793-5. PMC: 2700296. DOI: 10.1038/nmeth929. View

Hess S, Girirajan T, Mason M . Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006; 91(11):4258-72. PMC: 1635685. DOI: 10.1529/biophysj.106.091116. View

Sharonov A, Hochstrasser R . Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci U S A. 2006; 103(50):18911-6. PMC: 1748151. DOI: 10.1073/pnas.0609643104. View

Xu K, Zhong G, Zhuang X . Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science. 2012; 339(6118):452-6. PMC: 3815867. DOI: 10.1126/science.1232251. View

Szymborska A, de Marco A, Daigle N, Cordes V, Briggs J, Ellenberg J . Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science. 2013; 341(6146):655-8. DOI: 10.1126/science.1240672. View

Mund M, van der Beek J, Deschamps J, Dmitrieff S, Hoess P, Monster J . Systematic Nanoscale Analysis of Endocytosis Links Efficient Vesicle Formation to Patterned Actin Nucleation. Cell. 2018; 174(4):884-896.e17. PMC: 6086932. DOI: 10.1016/j.cell.2018.06.032. View

Shroder D, Lippert L, Goldman Y . Single molecule optical measurements of orientation and rotations of biological macromolecules. Methods Appl Fluoresc. 2017; 4(4):042004. PMC: 5308470. DOI: 10.1088/2050-6120/4/4/042004. View

Sosa H, Peterman E, Moerner W, Goldstein L . ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy. Nat Struct Biol. 2001; 8(6):540-4. DOI: 10.1038/88611. View

10.

Lippert L, Dadosh T, Hadden J, Karnawat V, Diroll B, Murray C . Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk. Proc Natl Acad Sci U S A. 2017; 114(23):E4564-E4573. PMC: 5468668. DOI: 10.1073/pnas.1620149114. View

11.

Backer A, Biebricher A, King G, Wuite G, Heller I, Peterman E . Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA. Sci Adv. 2019; 5(3):eaav1083. PMC: 6430628. DOI: 10.1126/sciadv.aav1083. View

12.

Ding T, Wu T, Mazidi H, Zhang O, Lew M . Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils. Optica. 2020; 7(6):602-607. PMC: 7440617. DOI: 10.1364/optica.388157. View

13.

Lu J, Mazidi H, Ding T, Zhang O, Lew M . Single-Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes. Angew Chem Int Ed Engl. 2020; 59(40):17572-17579. PMC: 7794097. DOI: 10.1002/anie.202006207. View

14.

Corrie J, Brandmeier B, Ferguson R, Trentham D, Kendrick-Jones J, Hopkins S . Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction. Nature. 1999; 400(6743):425-30. DOI: 10.1038/22704. View

15.

Forkey J, Quinlan M, Shaw M, Corrie J, Goldman Y . Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature. 2003; 422(6930):399-404. DOI: 10.1038/nature01529. View

16.

Chen C, Cui X, Beausang J, Zhang H, Farrell I, Cooperman B . Elongation factor G initiates translocation through a power stroke. Proc Natl Acad Sci U S A. 2016; 113(27):7515-20. PMC: 4941437. DOI: 10.1073/pnas.1602668113. View

17.

Backer A, Backlund M, Lew M, Moerner W . Single-molecule orientation measurements with a quadrated pupil. Opt Lett. 2013; 38(9):1521-3. PMC: 3991307. DOI: 10.1364/OL.38.001521. View

18.

Backer A, Moerner W . Extending single-molecule microscopy using optical Fourier processing. J Phys Chem B. 2014; 118(28):8313-29. PMC: 4317050. DOI: 10.1021/jp501778z. View

19.

Hafi N, Grunwald M, van den Heuvel L, Aspelmeier T, Chen J, Zagrebelsky M . Fluorescence nanoscopy by polarization modulation and polarization angle narrowing. Nat Methods. 2014; 11(5):579-84. DOI: 10.1038/nmeth.2919. View

20.

Zhanghao K, Chen L, Yang X, Wang M, Jing Z, Han H . Super-resolution dipole orientation mapping via polarization demodulation. Light Sci Appl. 2018; 5(10):e16166. PMC: 6059828. DOI: 10.1038/lsa.2016.166. View