Biology subjects

Reinkensmeier, L.

Publications and source records attributed to Reinkensmeier, L..

3 recordsLinked to original sources

bioRxiv2026

Cost-function Optimized Maximal Overlap Drift Estimation for Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) methods enable fluorescence imaging of biological specimens with nanometer-scale resolution. Although fluorophore localization precision is theoretically limited only by photon statistics, in practice the resolution of SMLM images is often degraded by physical drift of the sample and/or the microscope during data acquisition. At present, correcting this effect requires either specialized stabilization systems or computationally intensive post-processing, and established drift correction algorithms based on image cross-correlation suffer from limited temporal resolution. In this study we introduce COMET, a new method for SMLM drift estimation which achieves a substantially higher precision, accuracy, and temporal resolution compared with existing algorithmic approaches. We demonstrate that improved drift estimation translates directly into higher SMLM image resolution, limited by localization precision rather than drift artifacts. COMET is applicable to all types of SMLM data, operating directly on 2D or 3D localization datasets, and is readily integrated into analysis workflows. We benchmark its performance using both simulations and experiments, including STORM, MINFLUX, and Sequential OligoSTORM measurements, where long acquisition times make drift correction particularly challenging. COMET is published as an open-source, Python-based software project and is also available on open cloud-computing platforms.

biophysics↗

bioRxiv2025

Novel Laser Technology Enables 10x Faster SRS Imaging and Rapid Tuning in Biological Samples

Stimulated Raman Scattering (SRS) microscopy was developed for the label-free detection of molecular groups, addressing the speed limitations of spontaneous Raman microscopy. Standard SRS microscopy typically operates with laser sources at an 80 MHz repetition rate and a color-tuning speed of approximately 0.1 Hz to target different molecular groups. Here, we present a novel laser system that overcomes these speed limitations, achieving an order-of-magnitude improvement in both color-tuning and imaging speed. Our system features a reduced repetition rate of 40 MHz, enabling SRS imaging that is ten times faster than standard systems while maintaining the same average power at the sample. This is achieved through increased pulse energy and laser modulation at half the repetition rate. Furthermore, the system provides nearly ten times faster color-tuning across an extended range (660-1010 nm) by employing angle-tuning of nonlinear crystals instead of temperature-tuning. The improved performance is demonstrated in direct comparison with a standard SRS laser system, showcasing the potential for significantly enhanced imaging capabilities.

biophysics↗

bioRxiv2024

Near-infrared MINFLUX imaging enabled by suppression of fluorophore blinking

MINimal photon FLUXes (MINFLUX) offers super-resolution microscopy (SRM) with nanometer localization precision, with more relaxed fluorophore brightness and photostability requirements than for other SRM techniques. Nonetheless, low localization probabilities have been reported in several MINFLUX studies, and a broader use of less bright and photostable fluorophores, including near-infrared (NIR) fluorophores has been difficult to realize. In this work, we identified fluorophore blinking as a main cause of erroneous (and dismissed) fluorophore localizations in MINFLUX imaging and devised strategies to overcome these effects. We systematically studied the blinking/switching properties of cyanine fluorophores emitting in the far-red or NIR range, and over typical time scales ({micro}s-10ms), sample and excitation conditions used in MINFLUX imaging. By subsequent simulations of representative MINFLUX localization procedures, we found that trans-cis isomerization, and in particular photo-reduction of the fluorophores, can generate significant localization errors. However, these localization errors could be suppressed by balanced redox buffers and repetitive excitation beam scans. Implementing these strategies, and replacing the slower, intrinsic switching of the fluorophores needed for the localization by transient binding of fluorophore-labelled DNA strands to complementary DNA strands attached to the targets (DNA-PAINT), we could for the first time demonstrate NIR-MINFLUX imaging with nanometer localization precision. This work presents an overall strategy, where fluorophore blinking characterization and subsequent simulations make it possible to design optimal sample and excitation conditions, opening for NIR-MINFLUX imaging, as well as for a broader use of fluorophores in MINFLUX and related SRM studies.

biophysics↗