Single-Molecule Localization Microscopy and Tracking with a Fluorescent Mechanosensitive Probe DOI

Jimmy Maillard,

Ewa Grassin,

Ekaterina Bestsennaia

et al.

The Journal of Physical Chemistry B, Journal Year: 2024, Volume and Issue: 128(33), P. 7997 - 8006

Published: Aug. 9, 2024

A milestone in optical imaging of mechanical forces cells has been the development family flipper fluorescent probes able to report membrane tension noninvasively living through their fluorescence lifetime. The specifically designed Flipper-CF3 probe with an engineered inherent blinking mechanism was recently introduced for super-resolution microscopy lipid ordered membranes but too dim be detected disordered at single-molecule level (García-Calvo, J. Am. Chem. Soc. 2020, 142(28), 12034–12038). We show here that original and commercially available Flipper-TR is compatible based resolves both liquid giant unilamellar vesicles below diffraction limit. Single molecules were additionally tracked bilayers, enabling distinguish varying composition from diffusion coefficient probe. Differences brightness between originate steady-state absorption properties. general compatibility scaffold detection further shown experiments targetable derivatives.

Language: Английский

Design and Application of Fluorescent Probes to Detect Cellular Physical Microenvironments DOI

Junbao Ma,

Rui Sun,

Kaifu Xia

et al.

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(4), P. 1738 - 1861

Published: Feb. 14, 2024

The microenvironment is indispensable for functionality of various biomacromolecules, subcellular compartments, living cells, and organisms. In particular, physical properties within the biological could exert profound effects on both cellular physiology pathology, with parameters including polarity, viscosity, pH, other relevant factors. There a significant demand to directly visualize quantitatively measure fluctuation in spatiotemporal resolution. To satisfy this need, analytical methods based fluorescence probes offer great opportunities due facile, sensitive, dynamic detection that these molecules enable varying settings from vitro samples live animal models. Herein, we focus types small molecule fluorescent measurement microenvironment, mechanical force, temperature, electron potential. For each parameter, primarily describe chemical mechanisms underlying how are correlated changes signals. This review provides an overview perspective development environment, expand knowledge process, enrich diagnostic tools human diseases.

Language: Английский

Citations

106
Molecular Rotors: Fluorescent Sensors for Microviscosity and Conformation of Biomolecules DOI Creative Commons
Miguel Páez-Pérez, Marina K. Kuimova

Angewandte Chemie International Edition, Journal Year: 2023, Volume and Issue: 63(6)

Published: Oct. 19, 2023

Abstract The viscosity and crowding of biological environment are considered vital for the correct cellular function, alterations in these parameters known to underly a number pathologies including diabetes, malaria, cancer neurodegenerative diseases, name few. Over last decades, fluorescent molecular probes termed rotors proved extremely useful exploring viscosity, crowding, underlying interactions biologically relevant settings. In this review, we will discuss basic principles underpinning functionality review advances their use as sensors lipid order, protein conformation, temperature non‐canonical nucleic acid structures live cells other

Language: Английский

Citations

44
Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements DOI Creative Commons
Franziska Ragaller, Ellen Sjule,

Yagmur Balim Urem

et al.

The Journal of Physical Chemistry B, Journal Year: 2024, Volume and Issue: 128(9), P. 2154 - 2167

Published: Feb. 28, 2024

The structural diversity of different lipid species within the membrane defines its biophysical properties such as fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral in response to surrounding milieu, have greatly contributed our understanding properties. To realize full potential these probes avoid misinterpretation responses, a detailed investigation fluorescence characteristics environments is necessary. Here, we examined lifetime two newly developed order NR12S NR12A, alterations degree saturation, cholesterol content, double bond position configuration, phospholipid headgroup. As comparison, investigated sensitivity tension probe Flipper environments. Applying imaging microscopy (FLIM) both model membranes biological membranes, all distinguished phases by but exhibited sensitivities varying (e.g., cholesterol). While particularly sensitive NR12A lifetimes are moderately content acyl chains. Moreover, exhibit longer at emission wavelengths any complexity. This wavelength dependency results resolutions regions, highly relevant for FLIM data acquisition. Our provide valuable insights on how perform with highlight limitations.

Language: Английский

Citations

13
Fluorescent Solvatochromic Probes for Long‐Term Imaging of Lipid Order in Living Cells DOI Creative Commons

Takuya Tanaka,

Atsushi Matsumoto,

Andery S. Klymchenko

et al.

Advanced Science, Journal Year: 2024, Volume and Issue: 11(17)

Published: March 11, 2024

Abstract High‐resolution spatio‐temporal monitoring of the cell membrane lipid order provides visual insights into complex and sophisticated systems that control cellular physiological functions. Solvatochromic fluorescent probes are highly promising noninvasive visualization tools for identifying ordering microenvironment plasma microdomains. However, conventional probes, although capable structural analysis, lack necessary long‐term photostability required live imaging at level. Here, an ultra‐high‐light‐resistant solvatochromic fluorescence probe, 2‐ N , ‐diethylamino‐7‐(4‐methoxycarbonylphenyl)‐9,9‐dimethylfluorene (FπCM) is reported, which enables division. This probe its derivatives exhibit sufficient wavelengths, brightness, polarity responsiveness, low phototoxicity, remarkable under conditions compared to probes. Therefore, these have potential overcome limitations microscopy, particularly those associated with photobleaching. FπCM can serve as valuable elucidating mechanisms processes bio‐membrane

Language: Английский

Citations

11
Measuring plasma membrane fluidity using confocal microscopy DOI
Pablo Carravilla, Luca Andronico, Jan Schlegel

et al.

Nature Protocols, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 19, 2025

Language: Английский

Citations

1
Spectrally Resolved Single-Molecule Orientation Imaging Reveals a Direct Correspondence between the Polarity and Microviscosity Experienced by Nile Red in Supported Lipid Bilayer Membranes DOI

Aranyak Sarkar,

Jyotsna Bhatt Mitra, V. K. Sharma

et al.

The Journal of Physical Chemistry B, Journal Year: 2025, Volume and Issue: 129(9), P. 2380 - 2391

Published: Feb. 20, 2025

Molecular-level interactions among lipids, cholesterol, and water dictate the nanoscale membrane organization of lipid bilayers into liquid-ordered (Lo) liquid-disordered (Ld) phases, characterized by different polarities orders. Generally, solvatochromic dyes easily discriminate polarity difference between Lo Ld whereas molecular flippers rotors show distinct photophysics depending on order. Despite progress in single-molecule spectral imaging orientation mapping, direct experimental proof linking with microviscosity sensed same probe eludes us. Here, we demonstrate spectrally resolved localization microscopy to connect nanoscopic a bilayer its emission spectra, three-dimensional dipole orientation, rotational constraint offered local microenvironment highlight excellent correspondence order experienced probe. This technique has potential address heterogeneity dynamics, especially biology material sciences.

Language: Английский

Citations

1
Molecular Machines For The Control Of Transmembrane Transport DOI Creative Commons
Toby G. Johnson, Matthew J. Langton

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(50), P. 27167 - 27184

Published: Dec. 8, 2023

Nature embeds some of its molecular machinery, including ion pumps, within lipid bilayer membranes. This has inspired chemists to attempt develop synthetic analogues exploit membrane confinement and transmembrane potential gradients, much like their biological cousins. In this perspective, we outline the various strategies by which machines─molecular systems in a nanomechanical motion is exploited for function─have been designed be incorporated membranes utilized mediate transport. We survey machines spanning both switches motors, those that act as mobile carriers or are anchored membrane, mechanically interlocked molecules, examples activated response external stimuli.

Language: Английский

Citations

21
Dual Ratiometric Fluorescence Monitoring of Mechanical Polymer Chain Stretching and Subsequent Strain-Induced Crystallization DOI
K. SUGA, Takuya Yamakado, Shohei Saito

et al.

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(49), P. 26799 - 26809

Published: Dec. 5, 2023

Tracking the behavior of mechanochromic molecules provides valuable insights into force transmission and associated microstructural changes in soft materials under load. Herein, we report a dual ratiometric fluorescence (FL) analysis for monitoring both mechanical polymer chain stretching strain-induced crystallization (SIC) polymers. SIC has recently attracted renewed attention as an effective mechanism improving properties A polyurethane (PU) film incorporating trace dual-emissive flapping probe (N-FLAP, 0.008 wt %) exhibited blue-to-green FL spectral change low-stress region (<20 MPa), resulting from conformational planarization mechanically stretched chains. More importantly, at higher concentrations (∼0.65 %), PU showed second green to yellow during growth (20-65 MPa) due self-absorption scattered short wavelength region. The reversibility these was demonstrated by load-unload cycles. With results hand, degrees were quantitatively mapped monitored imaging based on different ratios (I525/I470 I525/I600). Simultaneous two mappings revealed spatiotemporal gap distribution SIC. combinational use is universal approach development matter physics.

Language: Английский

Citations

15
Fluorescent Probes Based on Charge and Proton Transfer for Probing Biomolecular Environment DOI Creative Commons
Vasyl G. Pivovarenko, Andrey S. Klymchenko

The Chemical Record, Journal Year: 2023, Volume and Issue: 24(2)

Published: Dec. 29, 2023

Abstract Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly lipids into biomembranes, interactions biomolecules imaging physiological state the cells. Here, we summarize major efforts in development based on two photophysical mechanisms: (i) an excited‐state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum a function environment hydration; (ii) proton (ESIPT), with particular focus 5‐membered cyclic systems, 3‐hydroxyflavones, because they exhibit dual sensitive environment. For both ICT ESIPT dyes, design biological applications are summarized. Thus, bearing amphiphilic anchors target lipid membranes report organization, while targeting ligands direct them specific organelles local The labels, amino acid nucleic analogues inserted enable monitoring membranes, proteins acids. While relatively simple robust environment‐sensitive probes, feature high information content due emission. They constitute powerful toolbox addressing multitude questions.

Language: Английский

Citations

14
Cell Membrane Tension Gradients, Membrane Flows, and Cellular Processes DOI
Qi Yan, Carolina Gomis‐Pérez, Erdem Karatekin

et al.

Physiology, Journal Year: 2024, Volume and Issue: 39(4), P. 231 - 245

Published: March 19, 2024

Cell membrane tension affects and is affected by many fundamental cellular processes, yet it poorly understood. Recent experiments show that can propagate at vastly different speeds in cell types, reflecting physiological adaptations. Here we briefly review the current knowledge about gradients, flows, their context.

Language: Английский

Citations

5