Biology subjects

Ruckebusch, C.

Publications and source records attributed to Ruckebusch, C..

3 recordsLinked to original sources

bioRxiv2023

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the usable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation. We use approach to simultaneously image four green photochromic fluorescent proteins at the full spatial resolution of the imaging. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/538742v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1f49247org.highwire.dtl.DTLVardef@1af85c2org.highwire.dtl.DTLVardef@768a92org.highwire.dtl.DTLVardef@83b81b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

A unifying perspective of the ultrafast photo-dynamics of Orange Carotenoid Protein from Synechocystis: peril of high-power excitation, existence of different S* states and influence of tagging

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of photo-activated form, OCPR. The most recent one suggests that three picosecond-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state which has the longest reported lifetime of a few to tens of picoseconds is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photo-dynamics of the OCP from Synechocystis PCC 6803, carried out using Visible-NIR femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photo-activation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S[~], living a few tens of picoseconds and formed only upon 470 nm excitation. Since neither the P1 quantum yield nor the photo-activation speed over hundreds of seconds vary under green and blue continuous irradiation, this S[~] species is unlikely to be involved in the photo-activation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on excited state photo-physical properties. Differences in spectral signatures and lifetimes of the different excited states were observed, at variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for careful consideration of the excitation power and His-tag position when comparing the photo-activation of different OCP variants, and beg to revisit the notion that S* is the precursor of photoactivated OCPR. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/474187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1064473org.highwire.dtl.DTLVardef@77e3c7org.highwire.dtl.DTLVardef@10b1e0eorg.highwire.dtl.DTLVardef@e24977_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Identifying microbial species by single-molecule DNA optical mapping and resampling statistics

Single molecule DNA mapping has the potential to serve as a powerful complement to high-throughput sequencing in metagenomic analysis. Offering longer read lengths and forgoing the need for complex library preparation and amplification, mapping stands to provide an unbiased view into the composition of complex viromes and/or microbiomes. To fully enable mapping-based metagenomics, sensitivity and specificity of DNA map analysis and identification need to be improved. Using detailed simulations and experimental data, we first demonstrate how fluorescence imaging of surface stretched, sequence specifically labeled DNA fragments can yield highly sensitive identification of targets. Secondly, a new analysis technique is introduced to increase specificity of the analysis, allowing even closely related species to be resolved. Thirdly, we show how an increase in resolution improves sensitivity. Finally, we demonstrate that these methods are capable of identifying species with long genomes such as bacteria with high sensitivity.

genomics↗