3D single-molecule super-resolution imaging of microfabricated fractal substrates for cell culture and self-referenced imaging DOI Creative Commons
Clément Cabriel, R. Margoth Córdova‐Castro, Erwin Berenschot

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Ноя. 10, 2023

Microstructures arrayed over a substrate have shown increasing interest due to their ability provide advanced 3D cellular models, which open new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by physical cues impact phenotype are not fully understood, hence necessity interrogate behavior at highest resolution. However, high-resolution optical imaging on such microstructured substrates remains challenging complexity, as well axial calibration issues. In this work, we address issue leveraging self-referenced characteristics of fractal-like structures, simultaneously modulate growth serve tools. To end, use multiscale SiO 2 consisting spatially octahedral features few micrometers hundreds nanometers. Through optimizations both structures conditions, demonstrate potential these tools super-resolution microscopy. We intrinsic nature perform lateral calibrations in single-molecule localization microscopy (SMLM) assess resolutions. then utilize platform bioimaging. As proof concept, cultivate human mesenchymal stem cells substrates, revealing very different patterns compared flat glass. Specifically, spatial distribution cytoskeleton proteins is vastly modified, with SMLM assessment.

Язык: Английский

Single-Molecule Fluorescence Lifetime Imaging Using Wide-Field and Confocal-Laser Scanning Microscopy: A Comparative Analysis DOI Creative Commons
Nazar Oleksiievets, Christeena Mathew, Jan Christoph Thiele

и другие.

Nano Letters, Год журнала: 2022, Номер 22(15), С. 6454 - 6461

Опубликована: Июль 6, 2022

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization (FL-SMLM). The synergy SMLM and imaging (FLIM) combines superior image resolution with lifetime information can be realized using two complementary experimental approaches: confocal-laser scanning (CLSM) or wide-field microscopy. Here, we systematically comprehensively compare these novel FL-SMLM approaches in different spectral regions. For FL-SMLM, use a commercial camera, for CLSM-based employ home-built system equipped rapid scan unit single-photon detector. We characterize performances systems localizing single emitters 3D by combining metal-induced energy transfer (MIET) along third dimension lifetime-based multiplexed bioimaging DNA-PAINT. Finally, discuss advantages disadvantages confocal provide practical advice on rational experiment design.

Язык: Английский

Процитировано

43
Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy DOI
Niels Radmacher, Oleksii Nevskyi, José Ignacio Gallea

и другие.

Nature Photonics, Год журнала: 2024, Номер 18(10), С. 1059 - 1066

Опубликована: Авг. 2, 2024

Язык: Английский

Процитировано

13
Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells DOI
Megan A. Steves, Changdong He, Ke Xu

и другие.

Annual Review of Physical Chemistry, Год журнала: 2024, Номер 75(1), С. 163 - 183

Опубликована: Фев. 16, 2024

By superlocalizing the positions of millions single molecules over many camera frames, a class super-resolution fluorescence microscopy methods known as single-molecule localization (SMLM) has revolutionized how we understand subcellular structures past decade. In this review, highlight emerging studies that transcend outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at levels. encoding/decoding high-dimensional information-such emission excitation spectra, motion, polarization, lifetime, beyond-for every molecule, mass accumulating these measurements for molecules, such multidimensional multifunctional approaches open new windows into intracellular architectures dynamics, well their underlying biophysical rules, far beyond diffraction limit.

Язык: Английский

Процитировано

12
Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy DOI Creative Commons
Jonas Zähringer,

Fiona Cole,

Johann Bohlen

и другие.

Light Science & Applications, Год журнала: 2023, Номер 12(1)

Опубликована: Март 10, 2023

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques fluorescence imaging. Here, we achieve by combining the 2D localization of pMINFLUX axial information graphene energy transfer (GET) and single-molecule switching DNA-PAINT. We demonstrate <2 nm precision in all 3 dimension reaching below 0.3 nm. In DNA-PAINT measurements, structural features, i.e., individual docking strands at distances nm, are directly resolved on DNA origami structures. GET represent particular synergetic combination for imaging near surface such as cell adhesion membrane complexes each photon used both information. Furthermore, introduce local PAINT (L-PAINT), which imager equipped an additional binding sequence upconcentration improving signal-to-background ratio speed clusters. L-PAINT demonstrated triangular structure 6 side lengths within seconds.

Язык: Английский

Процитировано

18
Local Water Content in Polymer Gels Measured with Super‐Resolved Fluorescence Lifetime Imaging DOI Creative Commons
Sankar Jana, Oleksii Nevskyi, Hannah Höche

и другие.

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(10)

Опубликована: Янв. 2, 2024

Water molecules play an important role in the structure, function, and dynamics of (bio-) materials. A direct access to number water nanoscopic volumes can thus give new molecular insights into materials allow for fine-tuning their properties sophisticated applications. The determination local content has become possible by finding that H

Язык: Английский

Процитировано

8
The power of super-resolution microscopy in modern biomedical science DOI
Nan Sun, Yi Jia, Shiwei Bai

и другие.

Advances in Colloid and Interface Science, Год журнала: 2023, Номер 314, С. 102880 - 102880

Опубликована: Март 13, 2023

Язык: Английский

Процитировано

15
Measuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy DOI Creative Commons
Tao Chen, Narain Karedla, Jörg Enderlein

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Фев. 27, 2024

Abstract Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability various methods for quantifying membrane dynamics, accurately complex systems with rapid tiny such mitochondria, remains challenge. In this work, we present methodology that combines metal/graphene-induced energy transfer (MIET/GIET) fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations simultaneous spatiotemporal resolution approximately one nanometer microsecond. To validate technique resolution, measure bending undulations model membranes. Furthermore, demonstrate versatility applicability MIET/GIET-FCS studying diverse systems, including widely studied fluctuating system human red blood cells, well two unexplored pore-spanning membrane, mitochondrial inner/outer

Язык: Английский

Процитировано

6
3D Super-Resolution Fluorescence Imaging of Microgels DOI Creative Commons
Oleksii Nevskyi, Dominik Wöll

Annual Review of Physical Chemistry, Год журнала: 2023, Номер 74(1), С. 391 - 414

Опубликована: Фев. 8, 2023

Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these have been applied hydrogel nano- and microparticles, so-called or microgels. We outline which research questions on microgels could be addressed what new insights achieved. Studies of the morphology, shape, deformation microgels; their internal compartmentalization; cross-linker distribution polarity inside them; dynamics diffusion summarized. particular, abilities super-resolve structures in three dimensions boosted field also allowed researchers obtain impressive 3D images deformed Accessing information beyond localization, such as spectral lifetime properties correlative imaging combination data with other methods, shines light onto systems helps us understand complexity detail. Such future trends developments addressed.

Язык: Английский

Процитировано

12
Mechanistic Insight into the Thermal “Blueing” of Cyanine Dyes DOI Creative Commons

Aria Vahdani,

Mehdi Moemeni,

Daniel Holmes

и другие.

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(29), С. 19756 - 19767

Опубликована: Июль 11, 2024

In recent work to develop cyanine dyes with especially large Stokes shifts, we encountered a "blueing" reaction, in which the heptamethine dye Cy7 (IUPAC: 1,3,3-trimethyl-2-((1E,3E,5E)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium) undergoes shortening two-carbon steps form pentamethine (Cy5) and trimethine (Cy3) analogs. Each step blue-shifts resulting absorbance wavelength by ca. 100 nm. Though photochemical oxidative chain-shortening reactions had been noted previously, it is simple heating alone or amine bases that effects this unexpected net C2H2 excision. Explicit acetylene loss would be too endothermic merit consideration. Our mechanistic studies using 2H labeling, mass spectrometric NMR spectroscopic analyses, quantum chemical modeling point instead electrocyclic closure aromatization of chain forming Fischer's base FB (1,3,3-trimethyl-2-methyleneindoline), reactive carbon nucleophile initiates attack on their polymethine backbones. The byproduct cationic indolium species TMP 1,3,3 trimethyl-2-phenyl indolium).

Язык: Английский

Процитировано

5
Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins DOI Creative Commons
Lara Hauke, Sebastian Isbaner, Arindam Ghosh

и другие.

ACS Nano, Год журнала: 2023, Номер 17(9), С. 8242 - 8251

Опубликована: Март 30, 2023

Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, implementation for live-cell with fluorescent proteins still lacking. Here, we present applicability capabilities diverse cell types (adult human stem cells, osteo-sarcoma Dictyostelium discoideum cells), various (GFP, mScarlet, RFP, YPet). We show MIET axial mapping living cellular subcellular components across multiple time scales, from few milliseconds to hours, negligible phototoxic effects.

Язык: Английский

Процитировано

10