Biology subjects

Brinks, D.

Publications and source records attributed to Brinks, D..

8 recordsLinked to original sources

bioRxiv2025

Robustness and fidelity of voltage imaging analysis pipelines

Tracking neuronal voltage changes using fluorescent voltage indicators is rapidly reshaping neuroscientific research. Voltage imaging enables direct visualization of electrical signals from subcellular compartments to large-scale networks, yet requires sophisticated image-analysis procedures. Here, we present a comprehensive study of current voltage imaging analysis pipelines and discuss the experimental conditions for which they are most suitable. We compare strengths and limitations of these pipelines in motion correction, denoising and segmentation routines, and discuss how different signal processing strategies can influence data integrity and interpretation. Our results show that most real-time analyses require GPU-accelerated algorithms and that denoising prior to signal processing is needed for analysis of subcellular dynamics. We conclude that voltage imaging analyses needs to be tailored to different experimental settings: we propose a decision-tree to determine analysis strategies for diverse experimental conditions. Together, these insights pave the way for reproducible, high-fidelity voltage imaging studies.

neuroscience↗

bioRxiv2024

Exploring Bioelectricity with Ace2N-mNeon during Zebrafish Embryogenesis

Bioelectricity is a fundamental biophysical phenomenon present in all cells, playing a crucial role in embryogenesis by regulating processes such as neuronal signaling, pattern formation, and cancer suppression. Precise monitoring of bioelectric signals and their dynamic changes throughout development is vital for advancing our understanding of higher organisms. However, the lack of suitable techniques for mapping bioelectric signals during early development has greatly limited our ability to interpret these mechanisms. To address this challenge, we developed an Ace2N-mNeon expression library in zebrafish, which exhibits membrane localization from 4 hours post-fertilization to at least 5 days post- fertilization and broad expression across multiple cell types throughout development. We validated the use of this library for studying bioelectric changes via voltage imaging to record signals in neurons and cardiomyocytes at different development stages. Through this approach, we found evidence of synchronized neuronal activity during early embryogenesis and observed faster voltage dynamics in cardiomyocytes as development progressed. Our results show that the Ace2N-mNeon library is a valuable tool for developmental bioelectric studies supporting advanced techniques such as voltage imaging and fluorescence lifetime imaging (FLIM). These methods enable non-invasive, dynamic monitoring of bioelectric signals across diverse cell types throughout development, significantly surpassing the capabilities of current electrophysiological techniques.

developmental biology↗

bioRxiv2024

In Vivo Plasmonic Enhancement of the Photocycle Dynamics and Fluorescence of Genetically Encoded Voltage Indicators

Plasmonic nanoparticles are key components in nanophotonics1-11 and have applications in molecular detection and diagnostic platforms. 12-14 Coupling of dipoles to plasmonic antennas has allowed engineering of qualitatively altered behavior in isolated quantum systems, 8,15 but this promise has not been fulfilled in living systems, where the use of plasmonics is limited to tissue level applications of plasmonic particles as contrast agents16 or heat sources.17 Here we show that coupling to designed plasmonic nanoparticles can control the electrophysiological function of proteins in living cells. We designed nanostar geometries and achieved robust near-field coupling of these optimized nanoparticles to plasma membrane-localized Archaerhodopsin proteins. We enhanced the fluorescence of the coupled rhodopsins and increased their response speed to membrane voltage. We incorporated this plasmonic enhancement into a Markov chain photocycle model of the Archaerhodopsin mutant QuasAr6a, showing an increased fluorescence emission rate and manipulation of the protein dynamics through a combination of photocycle transition rate enhancements. These results represent the first scalable near-field coupling between plasmonic particles and fluorescent proteins in living cells. They show enhancement of protein function beyond what has been achievable through genetic engineering. This opens up a range of possibilities for engineering biological functionality through plasmonics.

biophysics↗

bioRxiv2023

Expanding the family of genetically encoded voltage indicators: Insights from heliorhodopsins

Genetically encoded voltage indicators (GEVIs), particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel GEVI candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530-560nm, wild type heliorhodopsin exhibits near infra-red fluorescence which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity and kinetics of wild type heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution.

biophysics↗

bioRxiv2021

Functional single cell selection and annotated profiling of dynamically changing cancer cells

A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGF{beta} induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

bioengineering↗

bioRxiv2021

Linearly polarized excitation enhances signals from fluorescent voltage indicators

Voltage imaging in cells requires high-speed recording of small fluorescent signals, often leading to low signal-to-noise ratios. Because voltage indicators are membrane-bound, their orientations are partially constrained by the plane of the membrane. We explored whether tuning the linear polarization of excitation light could enhance voltage indicator fluorescence. We tested a panel of dye and protein-based voltage indicators in mammalian cells. The dye BeRST1 showed a 73% increase in brightness between the least and most favorable polarizations. The protein-based reporter ASAP1 showed a 22% change in brightness, and QuasAr3 showed a 14% change in brightness. In very thin neurites expressing QuasAr3, improvements were anomalously large, with a 170% increase in brightness between polarization parallel vs perpendicular to the dendrite. Signal-to-noise ratios of optically recorded action potentials were increased by up to 50% in neurites expressing QuasAr3. These results demonstrate that polarization control can be a facile means to enhance signals from fluorescent voltage indicators, particularly in thin neurites or in high-background environments.

biophysics↗

bioRxiv2018

All-optical electrophysiology reveals brain-state dependent changes in hippocampal subthreshold dynamics and excitability

A technology to record membrane potential from multiple neurons, simultaneously, in behaving animals will have a transformative impact on neuroscience research1. Parallel recordings could reveal the subthreshold potentials and intercellular correlations that underlie network behavior2. Paired stimulation and recording can further reveal the input-output properties of individual cells or networks in the context of different brain states3. Genetically encoded voltage indicators are a promising tool for these purposes, but were so far limited to single-cell recordings with marginal signal to noise ratio (SNR) in vivo4-6. We developed improved near infrared voltage indicators, high speed microscopes and targeted gene expression schemes which enabled recordings of supra- and subthreshold voltage dynamics from multiple neurons simultaneously in mouse hippocampus, in vivo. The reporters revealed sub-cellular details of back-propagating action potentials, correlations in sub-threshold voltage between multiple cells, and changes in dynamics associated with transitions from resting to locomotion. In combination with optogenetic stimulation, the reporters revealed brain state-dependent changes in neuronal excitability, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behavior.

neuroscience↗

bioRxiv2017

Two-photon photoactivated voltage imaging in tissue with an Archaerhodopsin-derived reporter

Robust voltage imaging in tissue remains a technical challenge. Existing combinations of genetically encoded voltage indicators (GEVIs) and microscopy techniques cannot simultaneously achieve sufficiently high voltage sensitivity, background rejection, and time resolution for highresolution mapping of sub-cellular voltage dynamics in intact brain tissue. We developed a pooled high-throughput screening approach to identify Archaerhodopsin mutants with unusual photophysical properties. After screening ~105 cells, we identified a novel GEVI, NovArch, whose 1-photon near infrared fluorescence is reversibly enhanced by weak 2-photon excitation. Because the 2-photon excitation acts catalytically rather than stoichiometrically, high fluorescence signals, optical sectioning, and high time resolution are achieved simultaneously, at modest 2-photon laser power. We developed a microscopy system optimized for NovArch imaging in tissue. The combination of protein and optical engineering enhanced signal contrast sufficiently to enable optical mapping of back-propagating action potentials in dendrites in acute mouse brain slice.

neuroscience↗