Biology subjects

Chen, I. A.

Publications and source records attributed to Chen, I. A..

12 recordsLinked to original sources

bioRxiv2023

Widefield in vivo imaging system with two fluorescence and two reflectance channels, a single sCMOS detector, and shielded illumination

SignificanceWidefield microscopy of the entire dorsal part of mouse cerebral cortex enables large-scale ("mesoscopic") imaging of different aspects of neuronal activity with spectrally compatible fluorescent indicators as well as hemodynamics via oxy- and deoxyhemoglobin absorption. Versatile and cost-effective imaging systems are needed for large-scale, color-multiplexed imaging of multiple fluorescent and intrinsic contrasts. AimDevelop a system for mesoscopic imaging of two fluorescent and two reflectance channels. ApproachExcitation of red and green fluorescence is achieved through epi-illumination. Hemoglobin absorption imaging is achieved using 525- and 625-nm LEDs positioned around the objective lens. An aluminum hemisphere placed between objective and cranial window provides diffuse illumination of the brain. Signals are recorded sequentially by a single sCMOS detector. ResultsWe demonstrate performance of our imaging system by recording large-scale spontaneous and stimulus-evoked neuronal, cholinergic, and hemodynamic activity in awake head-fixed mice with a curved "crystal skull" window expressing the red calcium indicator jRGECO1a and the green acetylcholine sensor GRABACh3.0. Shielding of illumination light through the aluminum hemisphere enables concurrent recording of pupil diameter changes. ConclusionsOur widefield microscope design with single camera can be used to acquire multiple aspects of brain physiology and is compatible with behavioral readouts of pupil diameter.

neuroscience↗

bioRxiv2023

Multi-Scale Label-free Human Brain Imaging with Integrated Serial Sectioning Polarization Sensitive Optical Coherence Tomography and Two-Photon Microscopy

The study of neurodegenerative processes in the human brain requires a comprehensive understanding of cytoarchitectonic, myeloarchitectonic, and vascular structures. Recent computational advances have enabled volumetric reconstruction of the human brain using thousands of stained slices, however, tissue distortions and loss resulting from standard histological processing have hindered deformation-free reconstruction of the human brain. The development of a multi-scale and volumetric human brain imaging technique that can measure intact brain structure would be a major technical advance. Here, we describe the development of integrated serial sectioning Polarization Sensitive Optical Coherence Tomography (PSOCT) and Two Photon Microscopy (2PM) to provide label-free multi-contrast imaging, including scattering, birefringence and autofluorescence of human brain tissue. We demonstrate that high-throughput reconstruction of 4x4x2cm3 sample blocks and simple registration of PSOCT and 2PM images enable comprehensive analysis of myelin content, vascular structure, and cellular information. We show that 2 m in-plane resolution 2PM images provide microscopic validation and enrichment of the cellular information provided by the PSOCT optical property maps on the same sample, revealing the sophisticated capillary networks and lipofuscin filled cell bodies across the cortical layers. Our method is applicable to the study of a variety of pathological processes, including demyelination, cell loss, and microvascular changes in neurodegenerative diseases such as Alzheimers disease (AD) and Chronic Traumatic Encephalopathy (CTE).

neuroscience↗

bioRxiv2022

High Throughput Detection of Capillary Stalling Events with Bessel Beam Two-Photon Microscopy

Disruptions in capillary flow have the potential to drive pathology across numerous diseases. But our understanding of the temporal and spatial dynamics of these events are hindered by slow volumetric imaging rates and the reliance on laborious manual analysis to process data. To address the challenges of increasing volumetric imaging speed, we use a custom-built Bessel beam two-photon microscope for efficient volumetric imaging of the capillary network. We demonstrate its ability to continuously monitor roughly 200 capillaries for capillary flow stoppages (i.e. stalling events) at a frame rate of approximately 0.5 Hz and develop a semi-automated correlation-based approach for identifying these stalling events. We applied our system and algorithm in a photothrombotic model of stroke and show elevated levels of stalling 1-week post-stroke in regions both within and outside of the stroke region, demonstrating that stalling may have impacts on stroke recovery that extend past the acute stage.

neuroscience↗

bioRxiv2022

Measuring Capillary Flow Dynamics using Interlaced Two-Photon Volumetric Scanning

Two photon microscopy and optical coherence tomography (OCT) are two standard methods for measuring flow speeds of red blood cells in microvessels, particularly in animal models. However, traditional two photon microscopy lacks the depth of field to adequately capture the full volumetric complexity of the cerebral microvasculature and OCT lacks the specificity offered by fluorescent labeling. In addition, the traditional raster scanning technique utilized in both modalities requires a balance of image frame rate and field of view, which severely limits the study of RBC velocities in the microvascular network. Here, we overcome this by using a custom two photon system with an axicon based Bessel beam to obtain volumetric images of the microvascular network with fluorescent specificity. We combine this with a novel scan pattern that generates pairs of frames with short time delay sufficient for tracking red blood cell flow in capillaries. We track flow speed in 10 or more capillaries simultaneously at 1 Hz in a 237 m x 237m x 120 m volume and quantify both spatial and temporal variability in speed. We also demonstrate the ability to track flow speed changes around stalls in capillary flow.

bioengineering↗

bioRxiv2022

A genetically engineered phage-based nanomaterial for detecting bacteria with magnetic resonance imaging

The ability to noninvasively detect bacteria at any depth inside opaque tissues has important applications ranging from infection diagnostics to tracking therapeutic microbes in their mammalian host. Current examples of probes for detecting bacteria with strain-type specificity are largely based on optical dyes, which cannot be used to examine bacteria in deep tissues due to the physical limitation of light scattering. Here, we describe a new biomolecular probe for visualizing bacteria in a cell-type specific fashion using magnetic resonance imaging (MRI). The probe is based on a peptide that selectively binds manganese and is attached in high numbers to the capsid of filamentous phage. By genetically engineering phage particles to display this peptide, we are able to bring manganese ions to specific bacterial cells targeted by the phage, thereby producing MRI contrast. We show that this approach allows MRI-based detection of targeted E. coli strains while discriminating against non-target bacteria as well as mammalian cells. By engineering the phage coat to display a protein that targets cell surface receptors in V. cholerae, we further show that this approach can be applied to image other bacterial targets with MRI. Finally, as a preliminary example of in vivo applicability, we demonstrate MR imaging of phage-labeled V. cholerae cells implanted subcutaneously in mice. The nanomaterial developed here thus represents a path towards noninvasive detection and tracking of bacteria by combining the programmability of phage architecture with the ability to produce three- dimensional images of biological structures at any arbitrary depth with MRI.

bioengineering↗

bioRxiv2021

High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4x0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.

neuroscience↗

bioRxiv2021

A kinetic error filtering mechanism for enzyme-free copying of nucleic acid sequences

Accurate copying of nucleic acid sequences is essential for self-replicating systems. Modern cells achieve error ratios as low as 10-9 with sophisticated enzymes capable of kinetic proofreading. In contrast, experiments probing enzyme-free copying of RNA and DNA as potential prebiotic replication processes find error ratios on the order of 10%. Given this low intrinsic copying fidelity, plausible scenarios for the spontaneous emergence of molecular evolution require an accuracy-enhancing mechanism. Here, we study a kinetic error filtering scenario that dramatically boosts the likelihood of producing exact copies of nucleic acid sequences. The mechanism exploits the observation that initial errors in template-directed polymerization of both DNA and RNA are likely to trigger a cascade of consecutive errors and significantly stall downstream extension. We incorporate these characteristics into a mathematical model with experimentally estimated parameters, and leverage this model to probe to what extent accurate and faulty polymerization products can be kinetically discriminated. While limiting the time window for polymerization prevents completion of erroneous strands, resulting in a pool in which full-length products show an enhanced accuracy, this comes at the price of a concomitant reduction in yield. We show that this fidelity-yield trade-off can be circumvented via repeated copying attempts in cyclically varying environments such as the temperature cycles occurring naturally in the vicinity of hydrothermal systems. This setting could produce exact copies of sequences as long as 50mers within their lifetime, facilitating the emergence and maintenance of catalytically active oligonucleotides.

evolutionary biology↗

bioRxiv2021

Error minimization and specificity could emerge in a genetic code as by-products ofprebiotic evolution

The emergence of the genetic code was a major transition in the evolution from a prebiotic RNA world to the earliest modern cells1. A prominent feature of the standard genetic code is error minimization, or the tendency of mutations to be unusually conservative in preserving biophysical features of the amino acid2-6. While error minimization is often assumed to result from natural selection, it has also been speculated that error minimization may be a by-product of emergence of the genetic code3. During establishment of the genetic code in an RNA world, self-aminoacylating ribozymes would enforce the mapping of amino acids to anticodons. Here we show that expansion of the genetic code, through co-option of ribozymes for new substrates, could result in error minimization as an emergent property. Using self-aminoacylating ribozymes previously identified during an exhaustive search of sequence space7, we measured the activity of thousands of candidate ribozymes on alternative substrates (activated analogs for tryptophan, phenylalanine, leucine, isoleucine, valine, and methionine). Related ribozymes exhibited preferences for biophysically similar substrates, indicating that co-option of existing ribozymes to adopt additional amino acids into the genetic code would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate that by-products of the evolution and functional expansion of a ribozyme system could lead to adaptive properties of a genetic code. Such spandrels could thus underlie significant prebiotic developments.

biochemistry↗

bioRxiv2021

Scalable mapping of myelin and neuron density in the human brain with micrometer resolution

Optical Coherence Tomography (OCT) is an emerging 3D imaging technique that allows quantification of intrinsic optical properties such as scattering coefficient and back-scattering coefficient, and has proved useful in distinguishing delicate microstructures in the human brain. The origins of scattering in brain tissues are contributed by the myelin content, neuron size and density primarily; however, no quantitative relationships between them have been reported, which hampers the use of OCT in fundamental studies of architectonic areas in the human brain and the pathological evaluations of diseases. To date, histology remains the golden standard, which is prone to errors and can only work on a small number of subjects. Here, we demonstrate a novel method that uses serial sectioning OCT to quantitatively measure myelin content and neuron density in the human brain. We found that the scattering coefficient possesses a strong linear relationship with the myelin content across different regions of the human brain, while the neuron density serves as a secondary contribution that only slightly modulates the overall tissue scattering.

neuroscience↗

bioRxiv2021

Flexible Simultaneous Mesoscale Two-Photon Imaging of Neural Activity at High Speeds

Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales.

neuroscience↗

bioRxiv2021

Large-scale cellular-resolution imaging of neural activity in freely behaving mice

Miniaturized microscopes for head-mounted fluorescence imaging are powerful tools for visualizing neural activity during naturalistic behaviors, but the restricted field of view of first-generation miniscopes limits the size of neural populations accessible for imaging. Here we describe a novel miniaturized mesoscope offering cellular-resolution imaging over areas spanning several millimeters in freely moving mice. This system enables comprehensive visualization of activity across entire brain regions or interactions across areas.

neuroscience↗

bioRxiv2020

Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters

Characterization of genotype-phenotype relationships of genetically encoded molecules (e.g., ribozymes) requires accurate quantification of activity for a large set of molecules. Kinetic measurement using high-throughput sequencing (e.g., k-Seq) is an emerging assay applicable in various domains that potentially scales up measurement throughput to 105 ~ 106 unique sequences. However, technical challenges introduced by sequence heterogeneity and DNA sequencing must be understood to realize the utility and limitations of such assays. We characterized the k-Seq method in terms of model identifiability, effects of sequencing error, accuracy and precision using simulated datasets and experimental data from a variant pool constructed from previously identified ribozymes. Relative abundance, kinetic coefficients, and measurement noise were found to affect the measurement of each sequence. We introduced bootstrapping to robustly quantify the uncertainty in estimating model parameters and proposed interpretable metrics to quantify model identifiability. These efforts enabled the rigorous reporting of data quality for individual sequences in k-Seq experiments. Critical experimental factors were examined, and general guidelines are proposed to maximize the number of sequences having precisely estimated and identifiable kinetic coefficients from k-Seq data. Practices analogous to those laid out here could be applied to improve the rigor of similar sequencing-based assays.

biochemistry↗