Direct optical measurement of intramolecular distances with angstrom precision DOI
Steffen J. Sahl, Jessica Matthias, Kaushik Inamdar

et al.

Science, Journal Year: 2024, Volume and Issue: 386(6718), P. 180 - 187

Published: Oct. 10, 2024

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative Förster resonance energy transfer (FRET) microscopy for decades. In this work, we show that MINFLUX fluorescence nanoscopy measures intramolecular down to 1 nanometer—and in planar projections angstrom—directly, linearly, and with angstrom precision. Our method was validated by quantifying well-characterized 1- 10-nanometer polypeptides proteins. Moreover, visualized orientations immunoglobulin applied human cells, revealed specific configurations a histidine kinase PAS domain dimer. results open door examining proximities interactions direct position measurements at intramacromolecular scale.

Language: Английский

MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells DOI
Klaus Gwosch, Jasmin K. Pape, Francisco Balzarotti

et al.

Nature Methods, Journal Year: 2020, Volume and Issue: 17(2), P. 217 - 224

Published: Jan. 13, 2020

Language: Английский

Citations

509
MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope DOI Creative Commons
Roman Schmidt, Tobias Weihs, Christian A. Wurm

et al.

Nature Communications, Journal Year: 2021, Volume and Issue: 12(1)

Published: March 5, 2021

Abstract The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising question method’s general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1–3 nm in three dimensions, rendering molecule-scale widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering stabilization locks sample position sub-nanometer precision respect stand, ensure nanometer-precise accurate real-time localization individually activated fluorophores. In our imaging cell- neurobiological samples, ~800 detected photons suffice 2.2 nm, whereas ~2500 yield precisions <1 (standard deviation). We further demonstrate 3D ~2.4 focal plane ~1.9 along optic axis. Localizing <20 within ~100 µs, establish this spatio-temporal single fluorophore tracking apply it diffusion labeled lipids lipid-bilayer model membranes.

Language: Английский

Citations

211
Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems DOI
Daniel J. Müller, Andra C. Dumitru, Cristina Lo Giudice

et al.

Chemical Reviews, Journal Year: 2020, Volume and Issue: 121(19), P. 11701 - 11725

Published: Nov. 9, 2020

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into nanoscopic laboratory to directly observe and chemically characterize molecular cell biological systems under physiological conditions. Here, we review that have established AFM as an analytical tool quantify native from micro- nanoscale. Native include living tissues, cells, cellular components such single or complexed proteins, nucleic acids, lipids, sugars. We showcase procedures customize chemical laboratories by functionalizing tips outline advantages limitations in applying different modes image, sense, manipulate biosystems at (sub)nanometer spatial millisecond temporal resolution. further discuss theoretical approaches extract kinetic thermodynamic parameters specific biomolecular interactions detected for bonds extend discussion multiple bonds. Finally, highlight potential combining with optical spectroscopy address full complexity tackle fundamental challenges life sciences.

Language: Английский

Citations

179
Fluorescent Nanoparticles for Super-Resolution Imaging DOI Creative Commons
Wei Li, Gabriele S. Kaminski Schierle, Bingfu Lei

et al.

Chemical Reviews, Journal Year: 2022, Volume and Issue: 122(15), P. 12495 - 12543

Published: June 27, 2022

Super-resolution imaging techniques that overcome the diffraction limit of light have gained wide popularity for visualizing cellular structures with nanometric resolution. Following pace hardware developments, availability new fluorescent probes superior properties is becoming ever more important. In this context, nanoparticles (NPs) attracted increasing attention as bright and photostable address many shortcomings traditional probes. The use NPs super-resolution a recent development provides focus current review. We give an overview different methods discuss their demands on NPs. then review in detail features, strengths, weaknesses each NP class to support these applications provide examples from utilization various biological systems. Moreover, we outlook future field opportunities material science multiplexed subcellular

Language: Английский

Citations

163
Super-Resolution Microscopy for Structural Cell Biology DOI
Sheng Liu, Philipp Hoess, Jonas Ries

et al.

Annual Review of Biophysics, Journal Year: 2022, Volume and Issue: 51(1), P. 301 - 326

Published: Feb. 4, 2022

Super-resolution microscopy techniques, and specifically single-molecule localization (SMLM), are approaching nanometer resolution inside cells thus have great potential to complement structural biology techniques such as electron for cell biology. In this review, we introduce the different flavors of super-resolution microscopy, with a special emphasis on SMLM MINFLUX (minimal photon flux). We summarize recent technical developments that pushed these localization-based scales review experimental conditions key obtaining data highest quality. Furthermore, give an overview analysis methods highlight studies used gain insights into biologically relevant molecular machines. Ultimately, our perspective what is needed push even further apply them investigating dynamic rearrangements in living cells.

Language: Английский

Citations

143
MINFLUX dissects the unimpeded walking of kinesin-1 DOI

Jan Otto Wolff,

Lukas Scheiderer, Tobias Engelhardt

et al.

Science, Journal Year: 2023, Volume and Issue: 379(6636), P. 1004 - 1010

Published: March 10, 2023

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 the protein, but requires detection of only about 20 photons from approximately 1-nanometer-sized fluorophore. Therefore, we were able study stepping motor kinesin-1 on microtubules at physiological adenosine-5′-triphosphate (ATP) concentrations. uncovered rotations stalk and heads load-free kinesin during showed ATP is taken a single head bound microtubule hydrolysis occurs when both are bound. Our results show quantifies (sub)millisecond conformational changes proteins minimal disturbance.

Language: Английский

Citations

118
Direct observation of motor protein stepping in living cells using MINFLUX DOI
Takahiro Deguchi, Malina K. Iwanski, Eva-Maria Schentarra

et al.

Science, Journal Year: 2023, Volume and Issue: 379(6636), P. 1010 - 1015

Published: March 10, 2023

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these have been challenging in living cells. Here, we developed live-cell tracking single fluorophores with nanometer spatial and millisecond temporal resolution two three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, resolved precise stepping motion motor protein kinesin-1 as it walked on microtubules Nanoscopic motors walking fixed cells also enabled us to resolve architecture microtubule cytoskeleton protofilament resolution.

Language: Английский

Citations

103
Molecular dynamics simulation of an entire cell DOI Creative Commons
Jan A. Stevens, Fabian Grünewald,

P. A. Marco van Tilburg

et al.

Frontiers in Chemistry, Journal Year: 2023, Volume and Issue: 11

Published: Jan. 18, 2023

The ultimate microscope, directed at a cell, would reveal the dynamics of all cell's components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on brink meeting this challenge. perspective, we show how an integrative approach can be employed model entire minimal JCVI-syn3A, full complexity. This step opens way interrogate spatio-temporal evolution molecular simulations, that extended other cell types in near future.

Language: Английский

Citations

89
Technological advances in super-resolution microscopy to study cellular processes DOI Creative Commons
Charles Bond, Adriana N. Santiago-Ruiz, Qing Tang

et al.

Molecular Cell, Journal Year: 2022, Volume and Issue: 82(2), P. 315 - 332

Published: Jan. 1, 2022

Language: Английский

Citations

80
DNA-PAINT MINFLUX nanoscopy DOI Creative Commons
Lynn M. Ostersehlt, Daniel C. Jans, Anna Wittek

et al.

Nature Methods, Journal Year: 2022, Volume and Issue: 19(9), P. 1072 - 1075

Published: Sept. 1, 2022

Abstract MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on–off switching process for single separation, initial MINFLUX implementations have been limited to two color channels. Here we show that can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding nanoscopy multiple molecular targets. Our method is exemplified three-color recordings of mitochondria in human cells.

Language: Английский

Citations

79