Biology subjects

Schreiter, E. R.

Publications and source records attributed to Schreiter, E. R..

At least 19 recordsLinked to original sources

bioRxiv2026

vFLIM: Machine Learning-enabled Light Sheet Fluorescence Lifetime Imaging

The lifetime of fluorescent molecules provides an orthogonal readout to fluorescence intensity, opening experimental possibilities of measuring changes in local molecular environments, mechanical tension, and metabolism, among other factors. These changes are best studied live and in vivo; however, limitations of slow imaging speeds, high phototoxicity, and increased data size and complexity have significantly impeded progress on this front. Here, we present a complete and transferable pipeline consisting of a light sheet FLIM microscope and an accompanying machine learning model for data processing that renders long-term and/or high-speed volumetric FLIM (vFLIM) tractable in living systems. We benchmark this pipeline across several biological use cases, model systems, lifetime ranges, and spatiotemporal scales, showcasing a suite of possibilities that our workflow enables. This comprehensive pipeline from imaging to analysis is a crucial step forward towards disseminating the power of live vFLIM to the broader bioimaging community.

bioengineering↗

bioRxiv2026

WHaloForce enables chemigenetic imaging of molecular tension in living cells and animals

Piconewton forces borne by individual proteins within complex assemblies underlie cell adhesion, migration, and tissue morphogenesis, yet remain difficult to image in living systems. Here, we introduce WHaloForce, a chemigenetic HaloTag-based tension sensor that converts force-dependent relief of tryptophan-mediated dye quenching into a fluorescence lifetime change. Optical tweezers revealed a switch-like unquenching transition near 5 pN, and the sensor responded reversibly to force changes in cells. WHaloForce enabled quantitative tension imaging of diverse force-bearing proteins (vinculin, E-cadherin, -catenin, and laminin) in mammalian cells, mouse tissue, and C. elegans. Bright synthetic dyes made tension measurements possible at endogenous expression levels. In C. elegans, vinculin and laminin showed opposite tension patterns between tissues, revealing distinct force-transmission routes through adhesions and the extracellular matrix. During ovulation, laminin tension accumulated over repeated stretch-relaxation cycles, scaling with cumulative loading history. WHaloForce thus offers a modular platform for imaging spatiotemporal tension patterns in living systems.

cell biology↗

bioRxiv2025

An expanded palette of bright and photostable organellar Ca2+ sensors

The use of fluorescent sensors for functional imaging has revolutionized the study of organellar Ca2+ signaling. However, understanding the dynamic interplay between intracellular Ca2+ sinks and sources has been hindered by the lack of bright, photostable, and multiplexed measurements in different organelles, limiting our ability to define how Ca2+ shapes cell physiology across fields of biology. Here we introduce a new toolkit of chemigenetic organellar Ca2+ indicators whose color is tunable by reconstituting their fluorescence with different exogenous rhodamine dye-ligands, which significantly expand the capacity for multiplexing organellar Ca2+ measurements. These sensors, which we named ER-HaloCaMP and Mito-HaloCaMP, are optimized to report Ca2+ dynamics in the endoplasmic reticulum (ER) and mitochondria of mammalian cells and neurons, and show significantly improved brightness, photostability and responsiveness when compared to current best-in-class alternatives. Using either red or far-red dye-ligands, both ER-HaloCaMP and Mito-HaloCaMP enable visualizing ER and mitochondrial Ca2+ dynamics in neuronal axons, a subcellular location that only contains a few ER tubules and small mitochondria, structural limitations that have impaired measurements with previous red sensors. To show the expanded multiplexing capacities of our toolkit, we measured interorganellar Ca2+ fluxes simultaneously in three different subcellular compartments in live cells, revealing that the amplitude of ER Ca2+ release controls the efficacy of ER-mitochondria Ca2+ coupling in a cooperative manner. Organellar HaloCaMPs enable also measuring Ca2+ dynamics in intact brain tissue from flies and rodents, demonstrating their versatility across biological models. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca2+ sensors, which will facilitate future studies of intracellular Ca2+ signaling across fields of biology in health and disease.

cell biology↗

bioRxiv2024

In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

neuroscience↗

bioRxiv2024

A novel rhodopsin-based voltage indicator for simultaneous two-photon optical recording with GCaMP in vivo

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

neuroscience↗

bioRxiv2024

Epigenetic repression of cFos supports sequential formation of distinct spatial memories

Expression of the immediate early gene cFos modifies the epigenetic landscape of activated neurons with downstream effects on synaptic plasticity. The production of cFos is inhibited by a long-lived isoform of another Fos family gene, {Delta}FosB. It has been speculated that this negative feedback mechanism may be critical for protecting episodic memories from being overwritten by new information. Here, we investigate the influence of {Delta}FosB inhibition on cFos expression and memory. Hippocampal neurons in slice culture produce more cFos on the first day of stimulation compared to identical stimulation on the following day. This downregulation affects all hippocampal subfields and requires histone deacetylation. Overexpression of {Delta}FosB in individual pyramidal neurons effectively suppresses cFos, indicating that accumulation of {Delta}FosB is the causal mechanism. Water maze training of mice over several days leads to accumulation of {Delta}FosB in granule cells of the dentate gyrus, but not in CA3 and CA1. Because the dentate gyrus is thought to support pattern separation and cognitive flexibility, we hypothesized that inhibiting the expression of {Delta}FosB would affect reversal learning, i.e., the ability to successively learn new platform locations in the water maze. The results indicate that pharmacological HDAC inhibition, which prevents cFos repression, impairs reversal learning, while learning and memory of the initial platform location remain unaffected. Our study supports the hypothesis that epigenetic mechanisms tightly regulate cFos expression in individual granule cells to orchestrate the formation of time-stamped memories.

neuroscience↗

bioRxiv2024

Synchronous ensembles of hippocampal CA1 pyramidal neurons associated with theta but not ripple oscillations during novel exploration

Synchronous neuronal ensembles play a pivotal role in the consolidation of long-term memory in the hippocampus. However, their organization during the acquisition of spatial memory remains less clear. In this study, we used neuronal population voltage imaging to investigate the synchronization patterns of CA1 pyramidal neuronal ensembles during the exploration of a new environment, a critical phase for spatial memory acquisition. We found synchronous ensembles comprising approximately 40% of CA1 pyramidal neurons, firing simultaneously in brief windows ([~]25ms) during immobility and locomotion in novel exploration. Notably, these synchronous ensembles were not associated with contralateral ripple oscillations but were instead phase-locked to theta waves recorded in the contralateral CA1 region. Moreover, the subthreshold membrane potentials of neurons exhibited coherent intracellular theta oscillations with a depolarizing peak at the moment of synchrony. Among newly formed place cells, pairs with more robust synchronization during locomotion displayed more distinct place-specific activities. These findings underscore the role of synchronous ensembles in coordinating place cells of different place fields.

neuroscience↗

bioRxiv2023

All-optical reporting of chloride ion driving force in the nervous system.

Ionic driving forces provide the net electromotive force for ion movement across receptors, channels, and transporters, and are a fundamental property of all cells. In the brain for example, fast synaptic inhibition is mediated by chloride permeable GABAA receptors, and single-cell intracellular recordings have been the only method for estimating driving forces across these receptors (DFGABAA). Here we present a new tool for quantifying inhibitory receptor driving force named ORCHID: all-Optical Reporting of CHloride Ion Driving force. We demonstrate ORCHIDs ability to provide accurate, high-throughput measurements of resting and dynamic DFGABAA from genetically targeted cell types over multiple timescales. ORCHID confirms theoretical predictions about the biophysical mechanisms that establish DFGABAA, reveals novel differences in DFGABAA between neurons and astrocytes, and affords the first in vivo measurements of intact DFGABAA. This work extends our understanding of inhibitory synaptic transmission and establishes a precedent for all-optical methods to assess ionic driving forces.

neuroscience↗

bioRxiv2023

A modular chemigenetic calcium indicator enables in vivo functional imaging with near-infrared light

Genetically encoded fluorescent calcium indicators have revolutionized neuroscience and other biological fields by allowing cellular-resolution recording of physiology during behavior. However, we currently lack bright, genetically targetable indicators in the near infrared that can be used in animals. Here, we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains that can be genetically targeted to specific cell populations. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several dye-ligands that efficiently label the central nervous system in animals. When bound to a near-infrared dye-ligand, WHaloCaMP1a is more than twice as bright as jGCaMP8s, and shows a 7x increase in fluorescence intensity and a 2.1 ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a with near-infrared fluorescence emission to image Ca2+ responses in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae, and to quantitate calcium concentration using fluorescence lifetime imaging microscopy (FLIM).

neuroscience↗

bioRxiv2022

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFRs nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

neuroscience↗

bioRxiv2021

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased {Delta}F/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

neuroscience↗

bioRxiv2021

Fast and sensitive GCaMP calcium indicators for imaging neural populations

Calcium imaging with protein-based indicators is widely used to follow neural activity in intact nervous systems. The popular GCaMP indicators are based on the calcium-binding protein calmodulin and the RS20 peptide. These sensors report neural activity at timescales much slower than electrical signaling, limited by their biophysical properties and trade-offs between sensitivity and speed. We used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators. The resulting jGCaMP8 sensors, based on calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (rise times, 2 ms) and still feature the highest sensitivity for neural activity reported for any protein-based sensor. jGCaMP8 sensors will allow tracking of larger populations of neurons on timescales relevant to neural computation.

neuroscience↗

bioRxiv2020

Far-red fluorescent genetically encoded calcium ion indicators

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ ({Delta}F/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.

neuroscience↗

bioRxiv2020

Bright and tunable far-red chemigenetic indicators

Functional imaging using fluorescent indicators has revolutionized biology but additional sensor scaffolds are needed to access properties such as bright, far-red emission. We introduce a new platform for chemigenetic fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. This approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in neurons.

molecular biology↗

bioRxiv2019

jYCaMP: An optimized calcium indicator for two-photon imaging at fiber laser wavelengths

State-of-the-art GFP-based calcium indicators do not undergo efficient two-photon excitation at wavelengths above 1000 nm, for which inexpensive and powerful industrial femtosecond lasers are available. Here we report jYCaMP1, a yellow variant of jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1000 nm and enables improved two-color calcium imaging with RFP-based indicators.

neuroscience↗

bioRxiv2019

rsCaMPARI: an erasable marker of neuronal activity

Identifying and comparing active neuron ensembles underlying complex behaviors is a key challenge in neuroscience. Recent tools such as CaMPARI have enabled the optical marking and selection of active neuron populations. However, CaMPARI photoconversion is permanent and irreversible, limiting its utility when multiple activity snapshots must be compared within the same sample. We sought to overcome these limitations by developing an erasable neuronal activity marker based on a reversibly switchable fluorescent protein. Here we introduce rsCaMPARI, a reversibly switchable calcium marker that enables spatiotemporally precise marking, erasing, and remarking of active neuron populations under brief, user-defined time windows of light exposure. rsCaMPARI photoswitching kinetics are modulated by calcium concentration when illuminating with blue light, and the fluorescence can be reset with violet light. We demonstrate the utility of rsCaMPARI for marking and remarking active neuron populations in freely-swimming zebrafish.

neuroscience↗

bioRxiv2019

A general approach to engineer positive-going eFRET voltage indicators

We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with \"positive-going\" fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

neuroscience↗

bioRxiv2019

Freeze-frame imaging of synaptic activity using SynTagMA

Information within the brain travels from neuron to neuron across synapses. At any given moment, only a few synapses within billions will be active and are thought to transmit key information about the environment, a behavior being executed or memory being recalled. Here we present SynTagMA, which marks active synapses within a ~2 s time window. Upon violet illumination, the genetically expressed tag converts from green to red fluorescence if bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed an analysis program that automatically identifies and tracks the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active synapses in vitro and in vivo.

neuroscience↗