Biology subjects

Rieger, B.

Publications and source records attributed to Rieger, B..

6 recordsLinked to original sources

bioRxiv2018

Photon count estimation in single-molecule localization microscopy

Recently, Franke, Sauer and van de Linde1 introduced a way to estimate the axial position of single-molecules (TRABI). To this end, they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found this photometric ratio to be around 0.7-0.8 close to focus and decreasing away from it. Here, we explain this reported but unexplained discrepancy and furthermore show that the photometric ratio as indicator for axial position is susceptible even to typical optical aberrations.

molecular biology↗

bioRxiv2018

F1FO ATP synthase responds to glycolysis inhibition by localization into the inner boundary membrane

Mitochondrial F1F0 ATP synthase is the key enzyme to fuel the cell with essential ATP. Strong indications exist that the respiratory chain and the ATP synthase are physically separated within cristae. How static this organization is, is largely unknown. Here, we investigated the effect of substrate restriction on mitochondrial respiration and the spatio-temporal organization of ATP synthase. By superresolution microscopy, the localization and mobility of single labelled mitochondrial ATP synthase was determined in live cells. We found, that the ATP synthase under oxidative respiration displayed a clear localization and confined mobility in cristae. Trajectories of individual ATP synthase proteins show a perpendicular course to the longitudinal axis of the respective mitochondrion, exactly following the ultrastructure of cristae. When substrate for TCA cycle and respiration was limited, a significant proportion of ATP synthase localized from cristae to the inner boundary membrane, and only less mobile ATP synthase remained in cristae. These observations showing the plasticity of the spatio-temporal organisation of ATP synthase can explain why ATP synthase show interactions with proteins in distinct mitochondrial subcompartments such as inner boundary membrane, cristae junctions and cristae.

cell biology↗

bioRxiv2018

Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities - 2D, astigmatic 3D, biplane 3D, and double helix 3D - and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

biophysics↗

bioRxiv2018

Photon yield enhancement of red fluorophores at cryogenic temperatures

Single Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 {middle dot} 106 to 11 {middle dot} 106 photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

biophysics↗

bioRxiv2017

Fluorescence polarization control for on-off switching of single molecules at cryogenic temperatures

Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which enable the interrogation of multiple molecular species at the same time in the same sample. Here we discuss the problems to be solved in the coming years to aim for higher resolution and describe what polarization depletion of fluorescence at cryogenic temperatures can contribute for fluorescence imaging of biological samples like whole cells.

biophysics↗

bioRxiv2017

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

biophysics↗