Washington University in St Louis

The Preston M. Green Department of
Electrical & Systems Engineering

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Overview

Super-resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules
ChemBioChem 19, 18 (2018)

Since the earliest invention of telescopes, microscopes, and eyeglasses, imaging systems have been designed to help humans visualize the world around us – big and small, near and far. These imaging systems collect the light reflected or emitted from an object and focus it onto our eyes or a camera. The design of these systems dictates that their images only contain two-dimensional (2D) information about an object and that their 2D images are blurred if the object is out of focus. We build imaging systems with new capabilities that surpass these shortcomings.

Super-resolution is a key feature of many of our imaging systems – the ability to overcome the resolution limit of wave physics, termed the diffraction limit, in order to visualize the nanoscale world. Read more about this technology on our resources page.

Measuring the orientation of single molecules

Concept

The orientation and rotations of fluorescent molecules have been used to study the movements of molecular motors along microtubules, the stretching and bending of DNA, the dynamics and composition of lipid membranes, and the spatial and temporal heterogeniety of various soft materials. However, current technologies cannot distinguish certain types of motion due to poor sensitivity. We design imaging systems to measure the orientation of single molecules by considering 1) how molecules are excited by polarized illumination, 2) the polarization of fluorescence light emitted by the molecules, and 3) the radiation pattern of the molecules. We are designing optimal point spread functions for measuring orientation.

The Tri-spot point spread function

Tri-spot PSF concept art Challenges when measuring orientational parameters of dipole-like emitters
  • Fluorophore brightness: need to resolve orientation and “wobble” of molecules without spreading their photons over too many measurements
  • Measurement degeneracy: using existing techniques, certain orientations produce similar images and cannot be resolved
We developed the Tri-spot point spread function for improved:
  • Temporal resolution: 3D orientational parameters of molecules in a large field of view are measured simultaneously using one camera frame
  • Orientation resolvability: each orientation produces a unique image
  • Near-optimal SNR: least number of measurements required to resolve all possible orientational second moments
  • High precision: optimized sensitivity towards all orientational parameters

Read

O. Zhang, J. Lu, T. Ding, and M. D. Lew, “Imaging the three-dimensional orientation and rotational mobility of fluorescent emitters using the Tri-spot point spread function,” Appl. Phys. Lett. 113, 031103 (2018). [Open Scholarship, Article, Summary PDF-266 KB]

Related articles

  1. M. D. Lew and W. E. Moerner, “Azimuthal polarization filtering for accurate, precise, and robust single-molecule localization microscopy,” Nano Lett. 14, 6407 (2014). [Article]
  2. M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, and W. E. Moerner, “The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging,” ChemPhysChem 15, 587 (2014). [Article]
  3. M. P. Backlund*, M. D. Lew*, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087 (2012). [Highlight in Nat. Methods, Article]

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Robust Statistical Estimation (RoSE) algorithm

Concept

RoSE demonstration

How can we estimate the likelihood that a fluorescent molecule is emitting light from each pixel within an object domain? And how can we estimate the likelihood that a molecule within a pixel belongs to an unknown target structure?

RoSE works for arbitrary 3D point spread functions (PSFs) and biological structures. It calculates the likelihood that each image pixel contains a molecule, and not background light, by leveraging spatial sparsity. Further, by analyzing blinking statistics, it can also calculate the confidence of each pixel in truly representing the target structure. RoSE thereby minimizes false localizations that cause bias and artifacts in super-resolution microscopy.

Read

H. Mazidi, J. Lu, A. Nehorai, and M. D. Lew, “Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy,” Sci. Rep. 8, 13133 (2018). [Article, Summary PDF-712 KB]

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Transient Amyloid Binding (TAB) super-resolution microscopy

Concept

Locations of blinking ThT molecules are combined to create a TAB super-resolution image of amyloid fibrils.

How can we visualize amyloid aggregation at nanometer resolution with minimum perturbation over extended time periods?

Amyloid aggregates are signatures of neurodegenerative disorders such as Alzheimer’s disease. We developed Transient Amyloid Binding (TAB) super-resolution microscopy to resolve amyloid structures using the standard probe, Thioflavin T (ThT), without the need for covalent modification or immunostaining of amyloids. Spontaneous binding and corresponding bursts of ThT fluorescence on amyloids are used to reconstruct super-resolution images of native amyloid structures.

Read

K. Spehar*, T. Ding*, Y. Sun, N. Kedia, J. Lu, G. R. Nahass, M. D. Lew, and J. Bieschke, “Super-Resolution Imaging of Amyloid Structures over Extended Times Using Transient Binding of Single Thioflavin T Molecules,” ChemBioChem 19, 1944 (2018). [Journal cover, The Source - Washington University, NSF Science360, EurekAlert!, Open Scholarship, Article, Summary PDF-408 KB]

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3D super-resolution microscopy

Concept

Two examples of rotating PSFs: (left) the corkscrew PSF and (right) the double-helix PSF.

We are investigating new ways of measuring the three-dimensional positions of objects from the two-dimensional images created by imaging systems. One powerful way to implement 3D imaging is through the use of engineered point spread functions (PSFs) – that is, a redesign of the imaging system such that a point emitter of light, like a single molecule, looks dramatically different from a normal focused spot. Rotating PSFs, or images that simply rotate as an emitter moves closer or further away from focus, are an efficient way to implement 3D super-resolution imaging. We are exploring the many possibilities for other PSF behaviors for 3D imaging.

Related articles

  1. M. D. Lew*, S. F. Lee*, J. L. Ptacin, M. K. Lee, R. J. Twieg, L. Shapiro, and W. E. Moerner, “Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus,” Proc. Natl. Acad. Sci. USA 108, E1102 (2011). [Article]
  2. M. D. Lew, S. F. Lee, M. Badieirostami, and W. E. Moerner, “Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects,” Opt. Lett. 36, 202 (2011). [Article]

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