GraFT: Graph Filtered Temporal Dictionary Learning for Functional Neural Imaging.

Optical imaging of calcium signals in the brain has enabled researchers to observe the activity of hundreds-to-thousands of individual neurons simultaneously. Current methods predominantly use morphological information, typically focusing on expected shapes of cell bodies, to better identify neurons in the field-of-view. The explicit shape constraints limit the applicability of automated cell identification to other important imaging scales with more complex morphologies, e.g., dendritic or widefield imaging. Specifically, fluorescing components may be broken up, incompletely found, or merged in ways that do not accurately describe the underlying neural activity. Here we present Graph Filtered Temporal Dictionary (GraFT), a new approach that frames the problem of isolating independent fluorescing components as a dictionary learning problem. Specifically, we focus on the time-traces-the main quantity used in scientific discovery-and learn a time trace dictionary with the spatial maps acting as the presence coefficients encoding which pixels the time-traces are active in. Furthermore, we present a novel graph filtering model which redefines connectivity between pixels in terms of their shared temporal activity, rather than spatial proximity. This model greatly eases the ability of our method to handle data with complex non-local spatial structure. We demonstrate important properties of our method, such as robustness to morphology, simultaneously detecting different neuronal types, and implicitly inferring number of neurons, on both synthetic data and real data examples. Specifically, we demonstrate applications of our method to calcium imaging both at the dendritic, somatic, and widefield scales.

Dynamic compartmental computations in tuft dendrites of layer 5 neurons during motor behavior.

Tuft dendrites of layer 5 pyramidal neurons form specialized compartments important for motor learning and performance, yet their computational capabilities remain unclear. Structural-functional mapping of the tuft tree from the motor cortex during motor tasks revealed two morphologically distinct populations of layer 5 pyramidal tract neurons (PTNs) that exhibit specific tuft computational properties. Early bifurcating and large nexus PTNs showed marked tuft functional compartmentalization, representing different motor variable combinations within and between their two tuft hemi-trees. By contrast, late bifurcating and smaller nexus PTNs showed synchronous tuft activation. Dendritic structure and dynamic recruitment of the N-methyl-d-aspartate (NMDA)-spiking mechanism explained the differential compartmentalization patterns. Our findings support a morphologically dependent framework for motor computations, in which independent amplification units can be combinatorically recruited to represent different motor sequences within the same tree.

Intrinsic Disruption of the M1 Cortical Network in a Mouse Model of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) disrupts motor performance by affecting the basal ganglia system. Yet, despite the critical position of the primary motor cortex in linking basal ganglia computations with motor performance, its contribution to motor disability in PD is largely unknown. The objective of this study was to characterize the role of the primary motor cortex in PD-related motor disability. METHODS: Two-photon calcium imaging and optogenetic stimulation of primary motor cortex neurons was done during performance of a dexterous reach-to-grasp motor task in control and 6-hydroxydopamine-induced PD mice. RESULTS: Experimental PD disrupted performance of the reach-to-grasp motor task and especially initiation of the task, which was partially restored by optogenetic activation of the primary motor cortex. Two-photon calcium imaging during task performance revealed experimental-PD affected the primary motor cortex in a cell-type-specific manner. It suppressed activation of output layer 5 pyramidal tract neurons, with greater effects on freeze versus nonfreeze trials. In contrast, it did not attenuate the initial movement-related activation response of layer 2/3 pyramidal neurons while diminishing the late inhibitory phase of their response. At the network level, experimental PD disrupted movement-related population dynamics of the layer 5 pyramidal tract network while almost not affecting the dynamics of the layer 2/3 neuronal population. It also disrupted short- and long-term robustness and stability of the pyramidal tract subnetwork, with reduced intertrial temporal accuracy and diminished reproducibility of motor parameter encoding and temporal recruitment of the output pyramidal tract neurons over repeated daily sessions. CONCLUSIONS: Experimental PD disrupts both external driving and intrinsic properties of the primary motor cortex. Motor disability in experimental PD results primarily from the inability to generate robust and stable output motor sequences in the parkinsonian primary motor cortex output layer 5 pyramidal tract subnetwork. © 2021 International Parkinson and Movement Disorder Society.

Whole-organism behavioral profiling reveals a role for dopamine in state-dependent motor program coupling in C. elegans.

Animal behaviors are commonly organized into long-lasting states that coordinately impact the generation of diverse motor outputs such as feeding, locomotion, and grooming. However, the neural mechanisms that coordinate these distinct motor programs remain poorly understood. Here, we examine how the distinct motor programs of the nematode C. elegans are coupled together across behavioral states. We describe a new imaging platform that permits automated, simultaneous quantification of each of the main C. elegans motor programs over hours or days. Analysis of these whole-organism behavioral profiles shows that the motor programs coordinately change as animals switch behavioral states. Utilizing genetics, optogenetics, and calcium imaging, we identify a new role for dopamine in coupling locomotion and egg-laying together across states. These results provide new insights into how the diverse motor programs throughout an organism are coordinated and suggest that neuromodulators like dopamine can couple motor circuits together in a state-dependent manner.

Animals generate many different motor programs (such as moving, feeding and grooming) that they can alter in response to internal needs and environmental cues. These motor programs are controlled by dedicated brain circuits that act on specific muscle groups. However, little is known about how organisms coordinate these different motor programs to ensure that their resulting behavior is coherent and appropriate to the situation. This is difficult to investigate in large organisms with complex nervous systems, but with 302 brain cells that control 143 muscle cells, the small worm Caenorhabditis elegans provides a good system to examine this question. Here, Cermak, Yu, Clark et al. devised imaging methods to record each type of motor program in C. elegans worms over long time periods, while also dissecting the underlying neural mechanisms that coordinate these motor programs. This constitutes one of the first efforts to capture and quantify all the behavioral outputs of an entire organism at once. The experiments also showed that dopamine ­ a messenger molecule in the brain ­ links the neural circuits that control two motor programs: movement and egg-laying. A specific type of high-speed movement activates brain cells that release dopamine, which then transmits this information to the egg-laying circuit. This means that worms lay most of their eggs whilst traveling at high speed through a food source, so that their progeny can be distributed across a nutritive environment. This work opens up the possibility to study how behaviors are coordinated at the level of the whole organism ­ a departure from the traditional way of focusing on how specific neural circuits generate specific behaviors. Ultimately, it will also be interesting to look at the role of dopamine in behavior coordination in a wide range of animals.

Rapid, Inexpensive Measurement of Synthetic Bacterial Community Composition by Sanger Sequencing of Amplicon Mixtures.

Synthetic bacterial communities are powerful tools for studying microbial ecology and evolution, as they enable rapid iteration between controlled laboratory experiments and theoretical modeling. However, their utility is hampered by the lack of fast, inexpensive, and accurate methods for quantifying bacterial community composition. Although next-generation amplicon sequencing can be very accurate, high costs (>$30 per sample) and turnaround times (>1 month) limit the nature and pace of experiments. Here, we quantify amplicon composition in synthetic bacterial communities through Sanger sequencing. We PCR amplify a universal marker gene, then we sequence this amplicon mixture in a single Sanger sequencing reaction. We then fit the "mixed" electropherogram with contributions from each community member as a linear combination of time-warped single-strain electropherograms, allowing us to estimate the fractional amplicon abundance of each strain within the community. This approach can provide results within one day and costs ∼$5 per sample.

Rapid and high-precision sizing of single particles using parallel suspended microchannel resonator arrays and deconvolution.

Measuring the size of micron-scale particles plays a central role in the biological sciences and in a wide range of industrial processes. A variety of size parameters, such as particle diameter, volume, and mass, can be measured using electrical and optical techniques. Suspended microchannel resonators (SMRs) are microfluidic devices that directly measure particle mass by detecting a shift in resonance frequency as particles flow through a resonating microcantilever beam. While these devices offer high precision for sizing particles by mass, throughput is fundamentally limited by the small dimensions of the resonator and the limited bandwidth with which changes in resonance frequency can be tracked. Here, we introduce two complementary technical advancements that vastly increase the throughput of SMRs. First, we describe a deconvolution-based approach for extracting mass measurements from resonance frequency data, which allows an SMR to accurately measure a particle's mass approximately 16-fold faster than previously possible, increasing throughput from 120 particles/min to 2000 particles/min for our devices. Second, we describe the design and operation of new devices containing up to 16 SMRs connected fluidically in parallel and operated simultaneously on the same chip, increasing throughput to approximately 6800 particles/min without significantly degrading precision. Finally, we estimate that future systems designed to combine both of these techniques could increase throughput by nearly 200-fold compared to previously described SMR devices, with throughput potentially as high as 24 000 particles/min. We envision that increasing the throughput of SMRs will broaden the range of applications for which mass-based particle sizing can be employed.

Modular Assembly of Polysaccharide-Degrading Marine Microbial Communities.

Understanding the principles that govern the assembly of microbial communities across earth's biomes is a major challenge in modern microbial ecology. This pursuit is complicated by the difficulties of mapping functional roles and interactions onto communities with immense taxonomic diversity and of identifying the scale at which microbes interact [1]. To address this challenge, here, we focused on the bacterial communities that colonize and degrade particulate organic matter in the ocean [2-4]. We show that the assembly of these communities can be simplified as a linear combination of functional modules. Using synthetic polysaccharide particles immersed in natural bacterioplankton assemblages [1, 5], we showed that successional particle colonization dynamics are driven by the interaction of two types of modules: a first type made of narrowly specialized primary degraders, whose dynamics are controlled by particle polysaccharide composition, and a second type containing substrate-independent taxa whose dynamics are controlled by interspecific interactions-in particular, cross-feeding via organic acids, amino acids, and other metabolic byproducts. We show that, as a consequence of this trophic structure, communities can assemble modularly-i.e., by a simple sum of substrate-specific primary degrader modules, one for each complex polysaccharide in the particle, connected to a single broad-niche range consumer module. Consistent with this model, a linear combination of the communities on single-polysaccharide particles accurately predicts community composition on mixed-polysaccharide particles. Our results suggest that the assembly of heterotrophic communities that degrade complex organic materials follows simple design principles that could be exploited to engineer heterotrophic microbiomes.

Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Linking single-cell measurements of mass, growth rate, and gene expression.

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.

Direct single-cell biomass estimates for marine bacteria via Archimedes' principle.

Microbes are an essential component of marine food webs and biogeochemical cycles, and therefore precise estimates of their biomass are of significant value. Here, we measured single-cell biomass distributions of isolates from several numerically abundant marine bacterial groups, including Pelagibacter (SAR11), Prochlorococcus and Vibrio using a microfluidic mass sensor known as a suspended microchannel resonator (SMR). We show that the SMR can provide biomass (dry mass) measurements for cells spanning more than two orders of magnitude and that these estimates are consistent with other independent measures. We find that Pelagibacterales strain HTCC1062 has a median biomass of 11.9±0.7 fg per cell, which is five- to twelve-fold smaller than the median Prochlorococcus cell's biomass (depending upon strain) and nearly 100-fold lower than that of rapidly growing V. splendidus strain 13B01. Knowing the biomass contributions from various taxonomic groups will provide more precise estimates of total marine biomass, aiding models of nutrient flux in the ocean.

Drug sensitivity of single cancer cells is predicted by changes in mass accumulation rate.

Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, the utility of current functional assays is limited, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. We found that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defined the drug sensitivity or resistance of glioblastoma and B-cell acute lymphocytic leukemia cells. MAR revealed heterogeneity in drug sensitivity not only between different tumors, but also within individual tumors and tumor-derived cell lines. MAR measurement predicted drug response using samples as small as 25 µl of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance in heterogeneous tumors.

High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays.

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes.

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores.

UNLABELLED: We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability. IMPORTANCE: Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance.

High-speed multiple-mode mass-sensing resolves dynamic nanoscale mass distributions.

Simultaneously measuring multiple eigenmode frequencies of nanomechanical resonators can determine the position and mass of surface-adsorbed proteins, and could ultimately reveal the mass tomography of nanoscale analytes. However, existing measurement techniques are slow (<1 Hz bandwidth), limiting throughput and preventing use with resonators generating fast transient signals. Here we develop a general platform for independently and simultaneously oscillating multiple modes of mechanical resonators, enabling frequency measurements that can precisely track fast transient signals within a user-defined bandwidth that exceeds 500 Hz. We use this enhanced bandwidth to resolve signals from multiple nanoparticles flowing simultaneously through a suspended nanochannel resonator and show that four resonant modes are sufficient for determining their individual position and mass with an accuracy near 150 nm and 40 attograms throughout their 150-ms transit. We envision that our method can be readily extended to other systems to increase bandwidth, number of modes, or number of resonators.

Weighing nanoparticles in solution at the attogram scale.

Physical characterization of nanoparticles is required for a wide range of applications. Nanomechanical resonators can quantify the mass of individual particles with detection limits down to a single atom in vacuum. However, applications are limited because performance is severely degraded in solution. Suspended micro- and nanochannel resonators have opened up the possibility of achieving vacuum-level precision for samples in the aqueous environment and a noise equivalent mass resolution of 27 attograms in 1-kHz bandwidth was previously achieved by Lee et al. [(2010) Nano Lett 10(7):2537-2542]. Here, we report on a series of advancements that have improved the resolution by more than 30-fold, to 0.85 attograms in the same bandwidth, approaching the thermomechanical noise limit and enabling precise quantification of particles down to 10 nm with a throughput of more than 18,000 particles per hour. We demonstrate the potential of this capability by comparing the mass distributions of exosomes produced by different cell types and by characterizing the yield of self-assembled DNA nanoparticle structures.

Intracellular water exchange for measuring the dry mass, water mass and changes in chemical composition of living cells.

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

Characterizing deformability and surface friction of cancer cells.

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies.

Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ∼2 ng of protein from a Barrett's esophagus biopsy.

Inhibition of secreted phospholipases A2 by 2-oxoamides based on α-amino acids: Synthesis, in vitro evaluation and molecular docking calculations.

Group IIA secreted phospholipase A2 (GIIA sPLA2) is a member of the mammalian sPLA2 enzyme family and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based on α-amino acids and the in vitro evaluation against three secreted sPLA2s (GIIA, GV and GX) are described. The long chain 2-oxoamide GK126 based on the amino acid (S)-leucine displayed inhibition of human and mouse GIIA sPLA2s (IC50 300nM and 180nM, respectively). It also inhibited human GV sPLA2 with similar potency, while it did not inhibit human GX sPLA2. The elucidation of the stereoelectronic characteristics that affect the in vitro activity of these compounds was achieved by using a combination of simulated annealing to sample low-energy conformations before the docking procedure, and molecular docking calculations.

Two-dimensional capillary electrophoresis: capillary isoelectric focusing and capillary zone electrophoresis with laser-induced fluorescence detection.

CIEF and CZE are coupled with LIF detection to create an ultrasensitive 2-D separation method for proteins. In this method, two capillaries are joined through a buffer-filled interface. Separate power supplies control the potential at the injection end of the first capillary and at the interface; the detector is held at ground potential. Proteins are labeled with the fluorogenic reagent Chromeo P503, which preserves the isoelectric point of the labeled protein. The labeled proteins were mixed with ampholytes and injected into the first-dimension capillary. A focusing step was performed with the injection end of the capillary at high pH and the interface at low pH. To mobilize components, the interface was filled with a high pH buffer, which was compatible with the second-dimension separation. A fraction was transferred to the second-dimension capillary for separation. The process of fraction transfer and second dimension separation was repeated two dozen times. The separation produced a spot capacity of 125.