Biology subjects

Huebinger, J.

Publications and source records attributed to Huebinger, J..

4 recordsLinked to original sources

bioRxiv2026

Robust thermometry-imaging at sub-micrometer and millisecond-resolution by fluorescence lifetime microscopy allows for additional acquisition of multiple imaging channels

Temperature is a fundamental parameter governing all molecular processes, including those that define life. Fluorescence microscopy is a powerful tool to observe molecular processes in living systems in real time. Precise control and measurement of temperature during fluorescence microscopy is therefore essential. We present here a robust temperature measurement based on the excited-state lifetime of the widely available and relatively inexpensive fluorescent dye pentamethine cyanine (Cy5). The excited-state lifetime of Cy5 shows a monotonic decline in the measurement range of 0 {degrees}C - 80 {degrees}C. The measured dependency is linear until 39 {degrees}C and monoexponential above. The dependance of excited-state lifetime upon temperature is used to measure temperature up to a precision of 0.5 {degrees}C or less, a temporal resolution down to <1 millisecond and to resolve temperature gradients with spatial resolutions that are only diffraction-limited. The far-red excitation and emission of Cy5 leaves bandwidth to simultaneously measure at least 3 additional spectral channels in standard fluorescent microscopes simultaneously. We demonstrate determination of temperature during 4-color live-cell fluorescence microscopy for a temperature-controlled experiment. We also show its applicability in measuring temperature gradients and laser-induced sample heating such as during STED nanoscopy.

biophysics↗

bioRxiv2025

Determination of factors that allow cryogenic nanoscopy with high power illumination without devitrification

Cryogenic super-resolution fluorescence light microscopy, or nanoscopy, has been demonstrated to be useful to close the resolution gap in cryogenic correlative light and electron microscopy (CLEM). Importantly, under cryogenic conditions fundamental resolution barriers that are imposed by molecular motion and photobleaching on fluorescence microscopy are circumvented. The resolution gain is even higher in nanoscopic methods, due to higher obtainable resolution and slower acquisition speed, rendering cryo-nanoscopy promising even beyond CLEM. However, cryogenic nanoscopy is often limited by heating and devitrification of the sample through strong laser irradiation, with drastic differences in reported tolerable power densities. We therefore investigated the laser-induced heating in different setups for cryogenic nanoscopy by time-dependent finite-element simulations complemented with absorption measurements of mammalian cells. Laser-induced heating happened in milliseconds, precluding efficient sample preservation by most intermittent illuminations. Under moderate light densities used for single molecule localization microscopy, absorbance by mammalian cells was too weak to explain devitrification and heating is governed by absorption of supporting material. However, the much higher power densities used in stimulated emission depletion nanoscopy resulted in temperatures clearly above the devitrification temperature from absorption by cells alone, unless the sample was mounted on an efficient heat exchanger, such as a diamond.

cell biology↗

bioRxiv2022

RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

Spatially-organized interaction dynamics between proto-oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases (PTPs) determine EGFRs phosphorylation response to growth factors and thereby cellular behavior within developing tissues. We show here, that and how the coupling between EGFR and RPTP{gamma} activity leads to migratory signaling responses to very low, physiological growth factor stimuli while suppressing aberrant, spontaneous signaling. Single cell imaging of EGFR phosphorylation and PTP oxidation revealed that RPTP{gamma} fully suppresses spontaneous EGFR phosphorylation, while EGF-induced NADPH-oxidase activity enables promigratory signaling responses at the plasma membrane by H2O2-mediated oxidative inhibition of RPTP{gamma}s phosphatase activity. The EGF-dependent toggle switch dynamics between interacting EGFR monomers and RPTP{gamma} thereby enables autocatalytically amplified phosphorylation responses to very low, physiological, EGF levels even at sparse receptor expression. This signaling mechanism is distinct from the proliferative signaling stemming from liganded endosomal EGFR complexes at high growth factor concentrations. Accordingly, RPTP{gamma} knock-out results in spontaneous promigratory EGFR signaling but loss of proliferative signaling. We thereby provide evidence of RPTP{gamma}s suppressor function of oncogenic, promigratory EGFR-signaling from the plasma membrane.

systems biology↗

bioRxiv2018

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes along with long acquisition times and high phototoxicity. Physiological live cell imaging is therefore often unfeasible and chemical fixation is employed. However, fixation methods have not been rigorously reviewed to preserve patterns at the resolution at which they can be nowadays imaged. A key parameter for this is the time span until fixation is completed. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from the cells. Other small aldehydes like glyoxal or acrolein showed inferior results. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely antagonized by supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

cell biology↗