RESUMO
Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.
Assuntos
Microscopia de Fluorescência/métodos , Neurônios/metabolismo , Sondas de Ácido Nucleico/metabolismo , Oligonucleotídeos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Difusão , Proteína 4 Homóloga a Disks-Large/metabolismo , Camundongos , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Neurônios/citologia , Sondas de Ácido Nucleico/química , Oligonucleotídeos/química , Ratos Sprague-Dawley , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (â¼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence â¼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.
Assuntos
Genoma Bacteriano/genética , Genômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Sequenciamento Completo do Genoma/métodos , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Resistência Microbiana a Medicamentos/genética , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Biblioteca Gênica , Genômica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Microbiologia do Solo , Sequenciamento Completo do Genoma/instrumentaçãoRESUMO
Effective screening methodologies for cells are challenged by the divergent and heterogeneous nature of phenotypes inherent to stem cell cultures, particularly on engineered biomaterial surfaces. In this study, we showcase a high-content, confocal imaging-based methodology to parse single-cell phenotypes by quantifying organizational signatures of specific subcellular reporter proteins and applied this profiling approach to three human stem cell types (embryonic-human embryonic stem cell [hESC], induced pluripotent-induced pluripotent stem cell [iPSC], and mesenchymal-human mesenchymal stem cell [hMSC]). We demonstrate that this method could distinguish self-renewing subpopulations of hESCs and iPSCs from heterogeneous populations. This technique can also provide insights into how incremental changes in biomaterial properties, both physiochemical and mechanical, influence stem cell fates by parsing the organization of stem cell proteins. For example, hMSCs cultured on polymeric films with varying degrees of poly(ethylene glycol) to modulate osteogenic differentiation were parsed using high-content organization of the cytoskeletal protein F-actin. In addition, hMSCs cultured on a self-assembled monolayer platform featuring compositional gradients were screened and descriptors obtained to correlate substrate variations with adipogenic lineage commitment. Taken together, high-content imaging of structurally sensitive proteins can be used as a tool to identify stem cell phenotypes at the single-cell level across a diverse range of culture conditions and microenvironments.