Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns.
Lab Chip
; 18(24): 3840-3848, 2018 12 04.
Article
em En
| MEDLINE
| ID: mdl-30417906
ABSTRACT
Fluorescence light microscopy (FLM) is commonly used for localizing specific cellular and subcellular targets. Electron microscopy (EM), on the other hand, can reveal ultrastructural details of cellular architectures beyond the limit of optical resolution. Correlative light and electron microscopy (CLEM) that combines the two techniques has proven valuable in various cell biological applications that require both specificity and resolution. Here, we report an efficient and easy-to-use CLEM system, and its applications in studying neuronal synapses. The system utilizes patterned symbols to encode coordinates on micro-fabricated polydimethylsiloxane (PDMS) substrates, on which dissociated primary hippocampal neurons grow and form synaptic connections. After imaging and localizing specifically labeled synapses with FLM, samples are embedded in resin blocks and sectioned for EM analysis. The patterned symbols on PDMS substrates provide coordinate information, allowing efficient co-registration between FLM and EM images with high precision. A custom-developed software package achieves automated EM image collection, FLM/EM alignment, and EM navigation. With this CLEM system, we have obtained high quality electron tomograms of fluorescently labeled synapses along dendrites of hippocampal neurons and analyzed docking statistics of synaptic vesicles (SVs) in different subtypes of excitatory synapses. This technique provides an efficient approach to combine functional studies with ultrastructural analysis of heterogeneous neuronal synapses, as well as other subcellular structures in general.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Processamento de Imagem Assistida por Computador
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Microscopia Eletrônica
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Técnicas Citológicas
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Dimetilpolisiloxanos
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Microscopia de Fluorescência
Limite:
Animals
Idioma:
En
Revista:
Lab Chip
Ano de publicação:
2018
Tipo de documento:
Article