Issue 24, 2018

Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns

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.

Graphical abstract: Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns

Supplementary files

Article information

Article type
Paper
Submitted
06 jul 2018
Accepted
18 okt 2018
First published
12 nov 2018
This article is Open Access
Creative Commons BY-NC license

Lab Chip, 2018,18, 3840-3848

Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns

R. Sun, X. Chen, C. Yin, L. Qi, P. Lau, H. Han and G. Bi, Lab Chip, 2018, 18, 3840 DOI: 10.1039/C8LC00703A

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements