Biology subjects

Koyama, M.

Publications and source records attributed to Koyama, M..

18 recordsLinked to original sources

bioRxiv2025

A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

cell biology↗

bioRxiv2023

Multiplane HiLo microscopy with speckle illumination and non-local means denoising

SignificanceHiLo microscopy synthesizes an optically-sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations, but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without compromising spatial resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here 9 planes). AimOur goal is to perform fast, high contrast multiplane imaging with a conventional camera-based fluorescence microscope. ApproachMultiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. ResultsWe demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. ConclusionsMultiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast, opticallysectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

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

TEMPO: A system to sequentially label and genetically manipulate vertebrate cell lineages

During development, regulatory factors appear in a precise order to determine cell fates over time. To investigate complex tissue development, one should not just label cell lineages but further visualize and manipulate cells with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labelling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation/inactivation of reporters/effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification. One-Sentence SummaryGaining sequential genetic access to vertebrate cell lineages.

developmental biology↗

bioRxiv2020

Single-cell transcriptomics of allo-reactive CD4+ T cells over time reveals divergent fates during gut GVHD

Acute gastrointestinal Graft-versus-Host-Disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem-cell transplantation (alloSCT). It is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN{gamma}, IL-17A or GM-CSF, and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between T-helper states during priming in mesenteric lymph nodes (mLN) and effector function in the GI tract remain undefined at genome-scale. We used scRNA-seq and computational modelling to create an atlas of putative differentiation pathways during GVHD. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we identified an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high TCF1 expression. TCF1hi cells upregulated 4{beta}7 prior to gut migration and failed to express cytokines therein. Nevertheless, they demonstrated recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq revealed divergence of allo-reactive CD4+ T cells into quiescent and effector states during gut GVHD, reflecting putative heterogenous priming in vivo. These findings, the first at a single-cell level during GVHD over time, can now be used to interrogate T cell differentiation in patients undergoing alloSCT.

immunology↗

bioRxiv2020

High-throughput cellular-resolution synaptic connectivity mapping in vivo with concurrent two-photon optogenetics and volumetric Ca2+ imaging

The ability to measure synaptic connectivity and properties is essential for understanding neuronal circuits. However, existing methods that allow such measurements at cellular resolution are laborious and technically demanding. Here, we describe a system that allows such measurements in a high-throughput way by combining two-photon optogenetics and volumetric Ca2+ imaging with whole-cell recording. We reveal a circuit motif for generating fast undulatory locomotion in zebrafish.

neuroscience↗

bioRxiv2019

Associations between Processing Speed and Psychopathology in a Transdiagnostic, Pediatric Sample

ObjectiveThe present study sought to examine the relationships between processing speed (PS), mental health disorders, and learning disorders. Prior work has tended to explore relationships between PS deficits and individual diagnoses (i.e., anxiety, autism, ADHD, depressive) in isolation of one another, often relying on relatively modest sample sizes. In contrast, the present work simultaneously investigated associations between PS deficits and these diagnoses, along with specific learning disabilities (i.e., reading, math), in a large-scale, transdiagnostic, community self-referred sample.\n\nMethodA total of 843 children, ages 8-16 were included from the Healthy Brain Network (HBN) Biobank. Given the presence of four PS tasks in HBN, principal component analysis (PCA) was employed to create a composite measure that represented the shared variance of the four PS tasks, referred to as PC1. Intraclass correlation coefficient (ICC) between the four PS measures, as well as PC1, were calculated to assess reliability. We then used multiple linear regression models to assess specific relationships between PS deficits and psychiatric diagnoses.\n\nResultsICCs were moderate between WISC-V tasks (0.663), and relatively modest between NIH Toolbox Pattern Comparison and other PS scales (0.14-0.27). Regression analyses revealed specific significant relationships between PS and reading and math disabilities, ADHD-inattentive type (ADHD-I), and ADHD-combined type (ADHD-C). Secondary analyses accounting for inattention dimensionally diminished associations with ADHD-C, but not ADHD-I or specific learning disability subtypes. The present study did not find a significant relationship with Autism Spectrum Disorder after accounting for inattentive symptoms. Consistent with prior work, demographic variables, including sex, socioeconomic status, and motor control exhibited independent relationships with PC1 as well.\n\nDiscussionThis study provided a comprehensive examination of PS, mental health disorders, and learning disabilities through a transdiagnostic approach. Implications for understanding how PS interacts with a highly heterogeneous childhood sample, as well as the need for increased focus on detection of affected populations are discussed.

developmental biology↗

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

CLADES: a programmable sequence of reporters for lineage analysis

We present CLADES (Cell Lineage Access Driven by an Edition Sequence), a technology for cell lineage studies based on CRISPR/Cas9. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a pre-determined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, coupling birth order with reporter expression. This gives us temporal resolution of lineage development that can be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, marking each generation with the corresponding combination of reporters. CLADES thus offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.\n\nOne Sentence SummaryA sequence of reporters for lineage analysis

developmental biology↗

bioRxiv2019

In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {micro}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed [~]3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

bioengineering↗

bioRxiv2018

Unlimited genetic switches for cell-type specific manipulation

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions, as well as to deliver precise gene therapy. Transcriptome analyses have allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types have remained inaccessible to genetic manipulation. Here, we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches that each respond to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell type-specific manipulations and combinatorial genetic targeting. CaSSA is thus a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

developmental biology↗

bioRxiv2018

Bright and photostable chemigenetic indicators for extended in vivo voltage imaging

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, Voltron, that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 minutes of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

neuroscience↗

bioRxiv2018

Chronology-based architecture of descending circuits that underlie the development of locomotor repertoire after birth.

The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system. Little is known, however, of how nascent connections are organized to support such new behaviors alongside existing ones. To understand this, in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion. We found that new projections are continually layered laterally to existing neuropil, and give rise to distinct pathways that function in parallel to existing pathways. Across these chronologically layered pathways, the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics. Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animals behavioral repertoire.

neuroscience↗

bioRxiv2018

Logical design of oral glucose ingestion pattern minimizing blood glucose in humans

Excessive increase in blood glucose level after eating increases the risk of macroangiopathy, and a method for not increasing the postprandial blood glucose level is desired. However, a logical design method of the dietary ingestion pattern controlling the postprandial blood glucose 2 level has not yet been established. We constructed a mathematical model of blood glucose control by oral glucose ingestion in 3 healthy human subjects, used the model to predict an optimal glucose ingestion pattern, and showed that the optimal ingestion pattern minimized the peak value of blood glucose level. Subjects orally ingested 3 doses of glucose by bolus or over 2 hours, and blood glucose, insulin, C-peptide and incretins were measured for 4 hours. We constructed an ordinary differential equation model that reproduced the time course data of the blood glucose and blood hormone levels. Using the model, we predicted that intermittent ingestion 30 minutes apart was the optimal glucose ingestion patterns that minimized the peak value of blood glucose level. We confirmed with subjects that this intermittent pattern decreased the peak value of blood glucose level. This approach could be applied to design optimal dietary ingestion patterns.\n\nIn BriefAs a forward problem, we measured blood glucose and hormones in three human subjects after oral glucose ingestion and constructed a mathematical model of blood glucose control. As an inverse problem, we used the model to predict the optimal oral glucose ingestion pattern that minimized the peak value of blood glucose level, and validated the pattern with the subjects.\n\nHighlightsO_LIModeling blood glucose concentrations predicts an intermittent ingestion pattern is optimal\nC_LIO_LIHuman validation shows ingestion at 30-minute intervals limits peak blood glucose\nC_LIO_LIWe provide a strategy to design optimal dietary ingestion patterns\nC_LI

systems biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗

bioRxiv2017

50 Hz volumetric functional imaging with continuously adjustable depth of focus

Understanding how neural circuits control behavior requires monitoring a large population of neurons with high spatial resolution and volume rate. Here we report an axicon-based Bessel beam module with continuously adjustable depth of focus (CADoF), which turns frame rate into volume rate by extending the excitation focus in axial direction while maintaining high lateral resolutions. Cost-effective and compact, this CADoF Bessel module can be easily integrated into existing two-photon fluorescence microscopes. Simply translating one of the relay lenses along its optical axis enabled continuous adjustment of the focal length. We used this module to simultaneously monitor activity of spinal projection neurons extending over 60 m depth in larval zebrafish at 50 Hz volume rate with adjustable imaging thickness.

neuroscience↗

bioRxiv2017

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

biophysics↗

bioRxiv2017

Stochastic Protein Labeling Enables Long-term Single Molecule Observation In Vivo

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

biophysics↗