A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos

Abstract

†These authors contributed equally to this work. The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples. Zebrafish are an excellent model system for cell and developmental biology. In addition to the power of this vertebrate system for genetic screening (1,2), the transparency of the zebrafish embryo has made it an ideal system for light microscopy that has been used with full advantage to study morphogenetic processes during development. Genetic screens are now being increasingly complemented by reverse genetic approaches, particularly involving mor-pholino-based approaches (3) but more recently using a powerful zinc finger nuclease-based approach (4,5). Zebrafish embryos are also ideal for electron microscopic (EM) studies; one transverse section of a 72 h embryo is easily contained in a single EM section, yet can contain numerous different cell types and tissues. However, conventional methods of EM do not provide optimal preservation of all tissues and are usually incompatible with immunolabeling and visualization of expressed fluorescently tagged proteins. An exception to this is the Tokuyasu frozen sectioning method, but the absence of a resin (6), while providing excellent antigen detection, is less suitable for preservation of large fluid-filled structures, such as the notochord of the zebrafish embryo, a crucial structural and signalling structure. Until recently, most EM analysis of the zebrafish has been performed using standard techniques that involve chemical fixation. In this study, we aimed to develop a technique that avoided chemical fixation, which allowed us to use the power of green fluorescent protein (GFP) as a fluorescent marker for light microscopy, while also allowing us to correlate light microscopic observations with immunogold EM on the same sections. Ideally, this technique would also allow us to perform three-dimensional (3D) electron tomography on the same sections. We now describe a method for high-pressure freezing of the zebrafish embryo that fulfils these criteria. The described method provides improved ultrastructure over conventional methods. Most importantly, with this method, we show that GFP fluorescence is still retained after freeze substitution (FS) and Lowicryl embedding of high-pressure frozen (HPF) embryos. As well as the detection of GFP-labelled proteins , endogenous antigens ...

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

“…For future applications, the here presented workflow can be adapted to be used for other resin embedded samples, e.g. Lowicryls, which have already been used in correlative studies (Nixon et al ., 2009;Kukulski et al ., 2011; Lucas et al . 2012).…”

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

“…Further work using CLEM showed that vesicle budding appears to involve the assembly of clathrin adaptors (Skruzny et al, 2015) and the bending of a dynamic preassembled clathrin coat (Avinoam et al, 2015). Similar approaches of preserving the fluorescence signal have been used in CLEM of zebrafish embryos (Nixon et al, 2009), C. elegans (Watanabe et al, 2011) and mammalian cells (Paez-Segala et al, 2015;Peddie et al, 2014). CLEM can also be performed thanks to the development of bimodal probes, such as the mini singlet oxygen generator (miniSOG) (Shu et al, 2011) and proteins of the ascorbate peroxidase (APX) family, in particular APEX2 (Lam et al, 2015).…”

Novel Contrasting and Labeling Procedures for Correlative Microscopy of Thawed Cryosections

“…Although many advances have been made in sample preparation for correlative microscopy, most protocols require processing or staining of the specimen in between the transfer from the FM to the EM (Roessler et al 1991;Ren et al 2003;van Rijnsoever et al 2008;Watanabe et al 2011;Kopek et al 2012). Protocols yielding resin-embedded specimens that can be imaged both with FM and TEM were recently developed (Nixon et al 2009;Kukulski et al 2011;. However, correlative imaging of …”