Immuno correlative light and electron microscopy on tokuyasu cryosections

Research output: Contribution to journalArticleOther

12 Citations (Scopus)

Abstract

Finding a rare structure by electron microscopy is the equivalent of finding a needle in a haystack. Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.
Original languageEnglish
Pages (from-to)241 - 258
Number of pages18
JournalMethods in Cell Biology
Volume124
Publication statusPublished - 2014

Cite this

@article{5920d590a8c947b8b3d1f6b85dabaf7f,
title = "Immuno correlative light and electron microscopy on tokuyasu cryosections",
abstract = "Finding a rare structure by electron microscopy is the equivalent of finding a needle in a haystack. Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.",
author = "Oorschot, {Viola M} and Sztal, {Tamar Esther} and Bryson-Richardson, {Robert James} and Georg Ramm",
year = "2014",
language = "English",
volume = "124",
pages = "241 -- 258",
journal = "Methods in Cell Biology",
issn = "0091-679X",
publisher = "Elsevier",

}

Immuno correlative light and electron microscopy on tokuyasu cryosections. / Oorschot, Viola M; Sztal, Tamar Esther; Bryson-Richardson, Robert James; Ramm, Georg.

In: Methods in Cell Biology, Vol. 124, 2014, p. 241 - 258.

Research output: Contribution to journalArticleOther

TY - JOUR

T1 - Immuno correlative light and electron microscopy on tokuyasu cryosections

AU - Oorschot, Viola M

AU - Sztal, Tamar Esther

AU - Bryson-Richardson, Robert James

AU - Ramm, Georg

PY - 2014

Y1 - 2014

N2 - Finding a rare structure by electron microscopy is the equivalent of finding a needle in a haystack. Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.

AB - Finding a rare structure by electron microscopy is the equivalent of finding a needle in a haystack. Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.

UR - http://www.ncbi.nlm.nih.gov/pubmed/25287844

M3 - Article

VL - 124

SP - 241

EP - 258

JO - Methods in Cell Biology

JF - Methods in Cell Biology

SN - 0091-679X

ER -