Biology subjects

Stiel, A. C.

Publications and source records attributed to Stiel, A. C..

10 recordsLinked to original sources

bioRxiv2026

Mutagenesis study of a Bacteriophytochrome - insights for the development of labels for optical imaging.

Bacteriophytochromes (BphPs) find increasing interest as near-infrared (NIR) labels for imaging. Applications range from whole animal imaging to cell-to super-resolution microscopy. Here we present a comprehensive study of a BphP from Rhizobium etli (ReBphP) allowing mutant-based insights into BphP photophysics. This is complemented by QM/MM-optimized deep-learning structure predictions of ReBphP and variants in their photoswitched states, rationalizing the effects of variants. Pertaining to imaging, based on our study we identify a bright and far red-shifted BphP for imaging in mammalian cells as well as a fluorescent photoswitching BphP. Utilizing the latter, we introduce photoswitching fluorescence background suppression for in vivo whole animal fluorescence imaging achieving higher contrast over background than possible utilizing non-switching labels.

biochemistry↗

bioRxiv2024

A fluorescent biosensor for the visualization of agmatine

Agmatine is known to regulate various neurotransmitter systems, yet the underlying molecular mechanisms remain elusive. To visualize agmatine dynamics in cellular networks and thereby unravel its physiological functions, we developed a genetically encoded fluorescent agmatine biosensor (AGMsen) based on the periplasmic putrescine binding protein PotF. We first analyzed the agmatine binding properties of the PotF receptor module based on its crystal structure and introduced mutations into the binding pocket to favor agmatine binding over other biogenic amines. Validation of AGMsen functionality across different cell types, including primary neuronal cultures, demonstrates its ability of real-time agmatine visualization. Thus, the sensor can serve as a valuable tool to advance our understanding of agmatine distribution and dynamics, and shed light on their effect on neuronal functions in vivo.

biochemistry↗

bioRxiv2024

Macroscopic label-free biomedical imaging withshortwave infrared Raman scattering

Shortwave infrared (SWIR) imaging provides enhanced tissue penetration and reduced autofluorescence in clinical and pre-clinical applications. However, existing applications often lack the ability to probe chemical composition and molecular specificity without the need for contrast agents. Here, we present a SWIR imaging approach that visualizes spontaneous Raman scattering with remarkable chemical contrast deep within tissue across large fields of view. Our results demonstrate that Raman scattering overcomes autofluorescence as the predominant source of endogenous tissue background at illumination wavelengths as short as 892 nm. We highlight the versatility of SWIR Raman imaging through in vivo monitoring of whole-body tissue composition dynamics and non-invasive detection of fatty liver disease in mice, and identification of calcification and lipids in unfixed human atherosclerotic plaques. Moreover, our approach facilitates the visualization of nerves embedded in fatty tissue, a major advancement for surgical applications. With a simple wide-field setup orthogonal to fluorescence, SWIR Raman imaging holds promise for rapid adoption by clinicians and biologists. This technique opens new possibilities for contrast agent-free visualization of pathophysiology in whole animals and intraoperative imaging in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/597863v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@394289org.highwire.dtl.DTLVardef@13f3289org.highwire.dtl.DTLVardef@ee6f7org.highwire.dtl.DTLVardef@5d7399_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Non-invasive optoacoustic imaging visualizes exercise-induced dermal revascularization in obese mice

Microcirculatory dysfunction in dermal (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans may predict cardio-metabolic disease progression. In-vivo visualization and monitoring of microvascular remodeling in these tissues remains challenging. We compared performance of multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT of diet-induced obese (DIO) mice undergoing voluntary wheel running. MSOT quantitatively visualized lipid and hemoglobin contrast in fat depots at early stages of DIO. RSOM precisely visualizes microvasculature with quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. Combination of MSOT and RSOM precisely monitor microcirculatory dysfunction and intervention response in dWAT and scWAT of DIO mice. Our findings lay out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in cardio-metabolic disease.

bioengineering↗

bioRxiv2020

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice

The increasing worldwide prevalence of obesity, fatty liver diseases, as well as the emerging understanding of the importance of lipids in multi-faceted aspects of various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. In this study, we investigate the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver as a biomarker to evaluate the organs function and assess the severity of NAFLD. This study demonstrates MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

bioengineering↗

bioRxiv2020

Optoacoustic flow-cytometry with light scattering referencing

In analogy to the development of fluorescent proteins, innovative tools for screening optoacoustic cell labels could lead to tailored protein labels for OA, imparting novel ways to visualize biological structure and function. Optoacoustic imaging emerges towards a highly promising modality for life sciences and medical practise with advantageous capabilities such as great accessible depth, and 3D studying of living tissue. The development of novel labels with molecular specificity could significantly enhance the optoacoustic contrast, specificity, and sensitivity and allow optoacoustic to interrogate tissues not amenable to the fluorescence method. We report on an optoacoustic flow cytometer (OAFC) prototype, developed for screening optoacoustic reporter genes. The cytometer concurrently records light scattering for referencing purposes. Since recording light scattering is completely independent from OA, we believe it to be a more reliable referencing method than e.g. fluorescence or ultrasound-backscatter. Precise characterization of our OAFC prototype showcases its ability to optoacoustically characterize objects in-flow that are in the size range of single cells. We apply the OAFC to distinguish individual E. coli cells based on optoacoustic properties of their expressed chromoproteins read in-flow using microfluidic arrangements and achieved precisions over 90%. We discuss how the light scattering referenced OAFC method offers a critical step towards routine measurement of optoacoustic properties of single-cells and could pave the way for identifying genetically encoded optoacoustic reporters, by transferring working concepts of the fluorescence field.

biophysics↗

bioRxiv2020

Multiplexed whole animal imaging with reversibly switchable optoacoustic proteins

We describe two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria and tumors in the mouse body and brain. We developed machine learning-based software that allows commercial imaging systems to be used for temporal unmixed OA imaging, enabling its routine use in life sciences.

biochemistry↗

bioRxiv2020

Challenging a preconception: Optoacoustic spectrum differs from the absorption spectrum of proteins and dyes for molecular imaging

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the optical absorption spectra do not correlate with the optoacoustic spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

biophysics↗

bioRxiv2020

Ligand promiscuity in the tryptophan repressor - from structural understanding towards rational design

Receptors that promiscuously bind a range of ligands provide insights into how nature mediates affinity and biological functioning. Moreover, such receptors provide vantage points for the rational design of specific binding for biotechnological applications. Here we describe the molecular details of the ligand binding promiscuity of the well-known tryptophan repressor TrpR. We elucidated high-resolution structures of TrpR bound to the co-repressors 5-methyl-tryptophan and 5-methyl-tryptamine as well as the pseudo-repressors indole-3-propionic and indole-3-acetic acid. Furthermore, using isothermal titration calorimetry we procure the corresponding thermodynamic parameters. Together this data provides molecular explanations for the strongly varied affinities and biological effects of the ligands providing insights into how nature shapes specificity and affinity. Beyond this we use these insights to exemplarily showcase knowledge-based design of TrpR by swapping its specificity from its native ligand tryptophan to indole-3-acetic acid. Finally, we elucidate the structures of the variant bound to indole-3-acetic and indole-3-propionic acid to retrace our design rationale.

synthetic biology↗

bioRxiv2020

Design of a biosensor for direct visualisation of auxin

In plants, one of the most important regulative small molecules is indole-3-acetic acid (IAA) known as auxin. Its dynamic redistribution plays an essential role in virtually every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, the spatial and temporal distribution of auxin at cellular resolution could not be determined directly. Instead it has been inferred from visualisation of irreversible processes involving the endogenous auxin response machinery3-7. This detection system failed to record transient changes. Here we report on a genetically encoded biosensor for quantitative in vivo visualisation of auxin distributions. The sensor is based on the E. coli tryptophan repressor (TrpR)8 whose binding pocket was engineered for specific IAA binding and coupled to fluorescent proteins to employ FRET as readout. This sensor, unlike previous systems, enables direct monitoring of the fast uptake and clearance of auxin by individual cells in the plant as well as the graded spatial distribution along the root axis and its perturbation by transport inhibitors. Thus, our auxin sensor enables mapping of auxin concentrations at (sub)cellular resolution and their changes in time and space during plant life.

plant biology↗