RESUMO
The mechanics of microneedle insertion have thus far been studied in a limited manner. Previous work has focused on buckling and failure of microneedle devices, while providing little insight into skin deformation, puncture, and the final positioning of needle tips under full microneedle arrays. The current study aims to develop a numerical approach capable of evaluating deformation and puncture conditions for full microneedle array designs. The analysis included a series of finite element submodels used to calibrate the microneedle-epidermal interface for failure properties using traction-separation laws. The single needle model is validated using experimental data and imaging, including results from a customized nanoindentation procedure to measure loads and displacements during microneedle insertion. Upon validation, full microneedle arrays are implemented in a 3 D finite element model and a design framework is developed, allowing evaluation of different design variables (i.e. needle shape, material, spacing) with respect to outputs relevant to successful microneedle performance. Results from the model include skin deformation, force to puncture, penetration depth, and the punctured state at each microneedle tip. In addition to microneedle parameters, patient parameters such as subcutaneous tissue thickness are included to evaluate the sensitivity of different microneedle designs to expected patient and anatomical region variability.
Assuntos
Desenho de Equipamento , Análise de Elementos Finitos , Microinjeções , Agulhas , Pele , Humanos , Microinjeções/instrumentação , Microinjeções/métodos , Sistemas de Liberação de Medicamentos/instrumentação , PunçõesRESUMO
Despite its importance, there is a poor understanding of human injury tolerance to trauma generally, and more specifically understanding of the mechanics of skin penetration or laceration. The objective of this analysis is to determine the failure criteria that will allow the evaluation of the laceration risk of blunt-tipped edges within a computational modeling environment. An axisymmetric tissue finite element model was set up in Abaqus 2021 to match the experimental set-up from a previous study. The model simulated the pressing of penetrometer geometries into dermal tissue, and stress and strain outputs were evaluated at the experimental failure force. Two separate nonlinear hyperelastic material models were calibrated for the dermis to data from the literature (high and low stiffness models). For both the high-stiffness and low-stiffness skin models, the failure force appears to occur near a local maximum in the principal strain. All failures occurred after the maximum strain near or at the top surface is or above 59%, with mid-thickness strain at a similar level. The strain energy density is concentrated near the edge tip for each configuration, indicating highly localized material damage at the point of loading, and increases rapidly prior to the approximate failure force. As the edge is further compressed into the tissue, the stress triaxiality near the edge contacting point decreases towards zero. This study has identified general failure criteria for skin laceration which can be implemented in a computational model. A higher risk for laceration would be indicated with strain energy density larger than 60 mJ/mm3, dermal strain larger than 55%, and stress triaxiality below 0.1. These findings were largely insensitive to the dermal stiffness and broadly applicable across different indenter geometries. It is expected that this framework may be implemented to evaluate hazardous forces for product edges, interactions with robots, and interfaces with medical and drug delivery devices.
Assuntos
Lacerações , Humanos , Estresse Mecânico , Simulação por Computador , Dinâmica não Linear , Pele , Análise de Elementos Finitos , Fenômenos BiomecânicosRESUMO
Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.
Assuntos
Perfilação da Expressão Gênica/métodos , Imagem Molecular/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Espinhas Dendríticas , Feminino , Humanos , Camundongos , Córtex VisualRESUMO
Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.
Assuntos
Encéfalo/diagnóstico por imagem , Cálcio/metabolismo , Corpo Celular/patologia , Neurônios/patologia , Imagem Óptica/métodos , Animais , Artefatos , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio , Corpo Celular/metabolismo , Proteínas de Fluorescência Verde , Camundongos , Neurônios/metabolismo , Neurópilo , Peixe-ZebraRESUMO
In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.
RESUMO
We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.
Assuntos
Evolução Molecular Direcionada/métodos , Proteínas Luminescentes/química , Engenharia de Proteínas/métodos , Robótica , Peixe-Zebra/embriologia , Animais , Encéfalo/diagnóstico por imagem , Caenorhabditis elegans , Separação Celular , Feminino , Citometria de Fluxo , Fluorescência , Biblioteca Gênica , Genes Reporter , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Camundongos , Microscopia de Fluorescência , Neurônios/citologia , OptogenéticaRESUMO
We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by â¼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded â¼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens â¼4.5 × 4.5, or â¼20×, and enables â¼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.
Assuntos
Aumento da Imagem/métodos , Micromanipulação/métodos , Microscopia/métodos , Polímeros/química , Manejo de Espécimes/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 µl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.
RESUMO
A large array of neuroscientific techniques, including in vivo electrophysiology, two-photon imaging, optogenetics, lesions, and microdialysis, require access to the brain through the skull. Ideally, the necessary craniotomies could be performed in a repeatable and automated fashion, without damaging the underlying brain tissue. Here we report that when drilling through the skull a stereotypical increase in conductance can be observed when the drill bit passes through the skull base. We present an architecture for a robotic device that can perform this algorithm, along with two implementations--one based on homebuilt hardware and one based on commercially available hardware--that can automatically detect such changes and create large numbers of precise craniotomies, even in a single skull. We also show that this technique can be adapted to automatically drill cranial windows several millimeters in diameter. Such robots will not only be useful for helping neuroscientists perform both small and large craniotomies more reliably but can also be used to create precisely aligned arrays of craniotomies with stereotaxic registration to standard brain atlases that would be difficult to drill by hand.
Assuntos
Encéfalo/cirurgia , Sistemas Computacionais , Craniotomia/instrumentação , Craniotomia/métodos , Potenciais de Ação , Algoritmos , Animais , Encéfalo/fisiologia , Camundongos , Tomografia por Raios XRESUMO
High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of â¼700 µm × 700 µm × 200 µm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.
Assuntos
Cálcio/metabolismo , Imageamento Tridimensional/métodos , Microscopia/métodos , Neurônios/fisiologia , Animais , Caenorhabditis elegans , Sinalização do Cálcio , Larva/ultraestrutura , Microscopia de Fluorescência/métodos , Peixe-ZebraRESUMO
Quantitative PCR (qPCR) techniques have become invaluable, high-throughput tools to study gene expression. However, the need to measure gene expression patterns quickly and affordably, useful for applications such as stem cell biomanufacturing requiring real-time observation and control, has not been adequately met by rapid qPCR instrumentation to date. We report a reverse transcription, microfluidic qPCR system and its application to DNA and RNA amplification measurement. In the system, an environmental control fixture provides mechanical and thermal repeatability for an infrared laser to achieve both accurate and precise open-loop temperature control of 1 µl reaction volumes in a low-cost polymer microfluidic chip with concurrent fluorescence imaging. We have used this system to amplify serial dilutions of λ-phage DNA (10(5)-10(7) starting copies) and RNA transcripts from the GAPDH housekeeping gene (5.45 ng total mouse embryonic stem cell RNA) and measured associated standard curves, efficiency (57%), repeatability (~1 cycle threshold), melting curves, and specificity. This microfluidic qRT-PCR system offers a practical approach to rapid analysis (~1 h), combining the cost benefits of small reagent volumes with the simplicity of disposable polymer microchips and easy setup.
Assuntos
Bacteriófago lambda/genética , DNA Viral/análise , Técnicas Analíticas Microfluídicas/instrumentação , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Animais , DNA Viral/genética , Desenho de Equipamento , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Camundongos , Técnicas Analíticas Microfluídicas/economia , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 µL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 µL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3 × 10(5) copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening.
Assuntos
Primers do DNA/genética , DNA Viral/análise , DNA Viral/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Microquímica/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL-5 µL), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 µL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.