HPM live μ for a full CLEM workflow

被引：10

作者：

Heiligenstein, Xavier ^{[1
]}

De Beer, Marit ^{[2
,3
,4
]}

Heiligenstein, Jerome ^{[1
]}

Eyraud, Frederique ^{[5
]}

Manet, Laurent ^{[1
]}

Schmitt, Fabrice ^{[6
]}

Lamers, Edwin ^{[7
]}

Lindenau, Joerg ^{[8
]}

Lindert, Mariska Kea-te ^{[2
,3
]}

Salamero, Jean ^{[9
,10
]}

Raposo, Grace ^{[11
,12
]}

Sommerdijk, Nico ^{[2
,4
]}

Belle, Martin ^{[1
]}

Akiva, Anat ^{[2
,3
]}

机构：

[1] CryoCapCell, Le Kremlin Bicetre, France

[2] Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Electron Microscopy Ctr, Radboudumc Technol Ctr Microscopy,Med Ctr, Nijmegen, Netherlands

[3] Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Dept Cell Biol, Med Ctr, Nijmegen, Netherlands

[4] Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Dept Biochem, Med Ctr, Nijmegen, Netherlands

[5] ARTechnol, Aydat, France

[6] Carl Zeiss SAS, Marly le Roi, France

[7] Carl Zeiss BV, Breda, Netherlands

[8] Carl Zeiss Microscopy GmbH, Jena, Germany

[9] CNRS, SERPICO Inria Team, UMR 144, Paris, France

[10] Inst Curie, Natl Biol & Hlth Infrastruct France Bioimaging, Paris, France

[11] PSL Res Univ, CNRS, Inst Curie, Cell & Tissue Imaging Facil PICT IBiSA,UMR144, Paris, France

[12] PSL Res Univ, Inst Curie, CNRS, Struct & Membrane Compartments,UMR144, Paris, France

来源：

CORRELATIVE LIGHT AND ELECTRON MICROSCOPY IV | 2021年 / 162卷

基金：

欧洲研究理事会;

关键词：

RAPID FREEZING SPRF; ELECTRON-MICROSCOPY; CORRELATIVE LIGHT; CRYOELECTRON MICROSCOPY; EC-CLEM; FLUORESCENCE; CRYOFIXATION; FIXATION; SUPERRESOLUTION; ULTRASTRUCTURE;

D O I：

10.1016/bs.mcb.2020.10.022

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

With the development of advanced imaging methods that took place in the last decade, the spatial correlation of microscopic and spectroscopic information-known as multimodal imaging or correlative microscopy (CM)-has become a broadly applied technique to explore biological and biomedical materials at different length scales. Among the many different combinations of techniques, Correlative Light and Electron Microscopy (CLEM) has become the flagship of this revolution. Where light (mainly fluorescence) microscopy can be used directly for the live imaging of cells and tissues, for almost all applications, electron microscopy (EM) requires fixation of the biological materials. Although sample preparation for EM is traditionally done by chemical fixation and embedding in a resin, rapid cryogenic fixation (vitrification) has become a popular way to avoid the formation of artifacts related to the chemical fixation/embedding procedures. During vitrification, the water in the sample transforms into an amorphous ice, keeping the ultrastructure of the biological sample as close as possible to the native state. One immediate benefit of this cryo-arrest is the preservation of protein fluorescence, allowing multi-step multi-modal imaging techniques for CLEM. To minimize the delay separating live imaging from cryo-arrest, we developed a highpressure freezing (HPF) system directly coupled to a light microscope. We address the optimization of sample preservation and the time needed to capture a biological event, going from live imaging to cryo-arrest using HPF. To further explore the potential of cryo-fixation related to the forthcoming transition from imaging 2D (cell monolayers) to imaging 3D samples (tissue) and the associated importance of homogeneous deep vitrification, the HPF core technology has been revisited to allow easy modification of the environmental parameters during vitrification. Lastly, we will discuss the potential of our HPM within CLEM protocols especially for correlating live imaging using the Zeiss LSM900 with electron microscopy.

引用

页码：115 / 149

页数：35

共 57 条

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