Biology subjects

Sahl, S. J.

Publications and source records attributed to Sahl, S. J..

5 recordsLinked to original sources

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↗

bioRxiv2023

Direct optical measurement of intra-molecular distances down to the Angstrom scale

Optical investigations of nanometer distances between proteins, their subunits, or other biomolecules have been the exclusive prerogative of Forster Resonance Energy Transfer (FRET) microscopy for decades. Here we show that MINFLUX fluorescence nanoscopy measures intra-molecular distances down to 1 nm - and in planar projections down to 1 Angstrom - directly, linearly, and with Angstrom precision. Our method is validated by quantifying well-characterized 1-to-10 nm distances in polypeptides and proteins. Moreover, we visualize the orientations of immunoglobulin subunits and reveal specific configurations of a histidine kinase domain dimer. Our results open the door for examining proximities and interactions of macromolecules under physiological conditions.

biophysics↗

bioRxiv2022

Assessment of 3D MINFLUX data for quantitative structural biology in cells revisited

Prakash and Curd provide a re-analysis1 of individual datasets taken from our report2 demonstrating MINFLUX 3D imaging in cells. Their evaluation confirms the unique localization precision provided by MINFLUX2,3 featuring a standard deviation of {sigma} = 1-3 nm. We appreciate their confirmation and also welcome the opportunity to clarify their remaining points. The hitherto almost unconceivable 3D localization precision attained by MINFLUX is likely to hold the key to an all-optical dynamical structural biology.

biophysics↗

bioRxiv2021

Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

biophysics↗

bioRxiv2019

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells.\n\nSignificance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

biochemistry↗