We introduce an interferometric MINFLUX microscope that records protein movements with up to 1.7 nanometer per millisecond spatiotemporal precision. Such precision has previously required attaching disproportionately large beads to the protein, but MINFLUX requires the detection of only about 20 photons from an approximately 1-nanometer-sized fluorophore. Therefore, we were able to study the stepping of the motor protein kinesin-1 on microtubules at up to physiological adenosine-5'-triphosphate (ATP) concentrations. We uncovered rotations of the stalk and the heads of load-free kinesin during stepping and showed that ATP is taken up with a single head bound to the microtubule and that ATP hydrolysis occurs when both heads are bound. Our results show that MINFLUX quantifies (sub)millisecond conformational changes of proteins with minimal disturbance.
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Localization microscopy enables imaging with resolutions that surpass the conventional optical diffraction limit. Notably, the Maximally INFormative LUminescence eXcitation (MINFLUX) method achieves super-resolution by shaping the excitation point spread function (PSF) to minimize the required photon flux for a given precision. Various beam shapes have recently been proposed to improve localization efficiency, yet their optimality remains an open question.
View Article and Find Full Text PDFMINFLUX fluorescence nanoscopy in biological tissue.
Proc Natl Acad Sci U S A
December 2024
Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen 37077, Germany.
Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue sample, superresolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments.
View Article and Find Full Text PDFExpression, Purification, and In Vitro Analysis of Myosin.
Methods Mol Biol
December 2024
Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, UK.
Article Synopsis
- The chapter explains the process of expressing and purifying myosin, a type of cytoskeletal molecular motor, using an insect cell system.
- It details methods for characterizing the quality of the purified myosin through techniques like mass photometry and negative-stain electron microscopy (EM).
- Lastly, it describes how to conduct in vitro assays to observe how fluorescently labeled myosin moves along actin tracks and mentions adapting these assays for MINFLUX imaging.
Tracking Single Kinesin in Live Cells Using MINFLUX.
Methods Mol Biol
December 2024
European Molecular Biology Laboratory, Cell Biology and Biophysics, Heidelberg, Germany.
MINFLUX is a super-resolution fluorescence microscopy technique that enables single-molecule tracking in live cells at a single-nanometer spatial and sub-millisecond temporal resolution. This chapter describes a method for tracking fluorescently labeled human kinesin-1 in live cells using MINFLUX and analyzing kinesin stepping dynamics.
View Article and Find Full Text PDFA new acquisition framework for MINFLUX super-resolution microscopy is proposed, termed Vortex Interference MINFLUX (viMINFLUX). From our analysis, we showed that by utilizing vortex interference, the scan range and phase shift mutually modulate MINFLUX precision, resulting in a precision enhancement by a factor of two or more for the same scan range. We further showed that vortex interference can be extended to 3D imaging, whereby 3D viMINFLUX provides for nearly isotropic 3D precision, resulting in a two-fold improvement in lateral precision and a five-fold enhancement in axial precision compared to conventional 3D MINFLUX techniques.
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