Biology subjects

Held, M. O.

Publications and source records attributed to Held, M. O..

3 recordsLinked to original sources

bioRxiv2024

Array Detection Enables Large Localization Range for Simple and Robust MINFLUX

The MINFLUX concept significantly enhances the spatial resolution of single-molecule localization microscopy (SMLM) by overcoming the limit imposed by the fluorophores photon counts. Typical MINFLUX microscopes localize the target molecule by scanning a zero-intensity focus around the molecule in a circular trajectory, with smaller trajectory diameters yielding lower localization uncertainties for a given number of photons. Since this approach requires the molecule to be within the scanned trajectory, MINFLUX typically relies on a photon-demanding iterative scheme with decreasing trajectory diameters. Although the iterative procedure does not substantially reduce the photon efficiency of MINFLUX, this approach is prone to misplacements of the trajectory and increases the systems complexity. In this work, we introduce ISM-FLUX, a novel implementation of MINFLUX using image-scanning microscopy (ISM) with a single-photon avalanche diode (SPAD) array detector. ISM-FLUX provides precise MINFLUX localization within the trajectory while maintaining conventional photon-limited uncertainty outside it. The robustness of ISM-FLUX localization results in a larger localization range and greatly simplifies the architecture, which may facilitate broader adoption of MIN-FLUX.

bioengineering↗

bioRxiv2023

4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter

We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered (fluorescence) photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty < 0.4 nm3 in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.

biophysics↗

bioRxiv2023

4D Single-Particle Tracking with Asynchronous Read-Out SPAD-Array Detector

Single-particle tracking (SPT) techniques are essential for investigating the com-plex functions and interactions of individual, specifically labelled particles in biological environments. Many SPT techniques exist, each optimised towards a different balance between spatiotemporal resolution and range, technical com-plexity, and information content. This bargain is exemplified by the contrast between wide-field camera-based and real-time SPT approaches, with the latter being generally more advanced but at the cost of high complexity. Further-more, the fluorescence lifetime, a powerful tool for investigating the particles interactions and nano-environment, has yet to be measured consistently. To overcome these limitations, we propose a novel real-time three-dimensional SPT technique based on a hybrid approach. In our implementation, we equip a confocal laser-scanning microscope with an asynchronous read-out single-photon avalanche diode (SPAD) array detector and few other optics. Each sensitive detector element acts as a confocal pinhole, and the recorded intensity distribu-tion reflects the particles position in three dimensions relative to the excitation volume. This localization is used in a real-time feedback system to keep the par-ticle in the centre of the excitation volume. Importantly, as each pixel is an independent single-photon detector, SPT is combined with fluorescence lifetime measurement. Our system achieves a localization precision of up to 30 nm with 100 photons and microsecond time resolution, while also performing fluorescence lifetime mea-surements. First, we validated the technique by tracking fluorescent particles in artificial environments. Secondly, as further validation, we investigated the move-ment of lysosomes in living SK-N-BE cells and measured the fluorescence lifetime of the GFP marker expressed on a membrane protein. We observed an unprece-dented correlation between the changes in fluorescence lifetime and the motion state of the lysosomes. Thanks to its simplicity and the great momentum of confocal microscopy based on SPAD array detector, we expect that this implementation will open to many information-rich correlative imaging and tracking studies.

biophysics↗