Biology subjects

Leutenegger, M.

Publications and source records attributed to Leutenegger, M..

6 recordsLinked to original sources

bioRxiv2026

3D-MINSTED nanoscopy and protein tracking in densely labelled living cells

Investigating the movements and conformational changes of proteins in living cells is essential for understanding their function. The recently introduced fluorescence nanoscopy method called MINSTED has successfully tracked single fluorophore-labelled proteins, albeit in fixed cells and two dimensions only. Here we introduce a MINSTED setup for live-cell tracking of individual proteins in three dimensions (3D) with a localization precision {sigma} down to < 1 nm. Applied to the motor protein kinesin-1, our MINSTED nanoscope follows single proteins in 3D as they process along microtubules in 16 nm steps. Individual steps are resolved amid substantial intracellular background. The unique capability of the 3D stimulated emission depletion (STED) beam to carve out the signal of the protein label of interest enables efficient single-molecule investigations at hitherto unpractically high concentrations of labelled proteins and thus in crowded live-cell environments.

biophysics↗

bioRxiv2024

MINFLUX fluorescence nanoscopy in biological tissue

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 medium, super-resolution 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. Here, we investigated the performance of MINFLUX imaging for different synaptic targets and fluorescent labels in tissue sections of the mouse brain. Single fluorophores were localized with a precision of < 5 nm at up to 80 {micro}m sample depth. MINFLUX imaging in two color channels allowed to probe PSD95 localization relative to the spine head morphology, while also visualizing presynaptic VGlut clustering and AMPA receptor clustering at the post-synapse. Our two-dimensional (2D) and three-dimensional (3D) two-color MINFLUX results in tissue, with < 10 nm 3D fluorophore localization, open up new avenues to investigate protein distributions on the single-synapse level in fixed and living brain slices.

biophysics↗

bioRxiv2022

Simultaneous multicolor fluorescence imaging using duplication-based PSF engineering

We present a way to encode more information in fluorescence imaging by splitting the emission into copies of the original point-spread function (PSF), which offers broadband operation and compatibility with other PSF engineering modalities and existing analysis tools. We demonstrate the approach using the Circulator, an add-on that encodes the fluorophore emission band into the PSF, enabling simultaneous multicolor super-resolution and single-molecule microscopy using essentially the full field of view.

biophysics↗

bioRxiv2022

MINSTED nanoscopy enters the Angstrom localization range

We report all-optical, room-temperature localization of fluorophores with precision in the [A]ngstrom range. These precisions are attained in a STED microscope, by encircling the fluorophore with the low-intensity edge of the STED donut beam, while constantly increasing the absolute donut power. Individual fluorophores bound to a DNA strand are localized with{sigma} = 4.7 [A], corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Since our experiments yield a localization precision{sigma} = 2.3 [A], estimated for 10,000 detected photons, we anticipate that MINSTED will open up entirely new areas of application in the study of macromolecular complexes in cells.

biophysics↗

bioRxiv2020

MINSTED fluorescence localization and nanoscopy

We introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

biophysics↗

bioRxiv2020

A quantitative analysis of the interplay of environment, neighborhood and cell state in 3D spheroids

1Cells react to their microenvironment by integrating external stimuli into phenotypic decisions via an intracellular signaling network. Even cells with deregulated signaling can adapt to their environment. To analyze the interplay of environment, neighborhood, and cell state on phenotypic variability, we developed an experimental approach that enables multiplexed mass cytometric imaging to analyze up to 240 pooled spheroid microtissues. This system allowed us to quantify the contributions of environment, neighborhood, and intracellular state to phenotypic variability in spheroid cells. A linear model explained on average more than half of the variability of 34 markers across four cell lines and six growth conditions. We found that the contributions of cell-intrinsic and environmental factors are hierarchically interdependent. By overexpression of 51 signaling protein constructs in subsets of cells, we identified proteins that have cell-intrinsic and extrinsic effects, exemplifying how cell states depend on the cellular neighborhood in spheroid culture. Our study deconvolves factors influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, analytical principles, and rich multiplexed imaging datasets for future studies.

systems biology↗