Biology subjects

Stiel, A. C.

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

5 recordsLinked to original sources

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↗