Correlative light and electron microscopy. 2 by Thomas Mueller-Reichert, Paul Verkade

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By Thomas Mueller-Reichert, Paul Verkade

This new quantity of Methods in phone Biology seems at equipment for studying correlative mild and electron microscopy (CLEM). With CLEM, humans try and mix the benefits of either worlds, i.e. the dynamics info got by means of mild microscopy and the ultrastructure as supplied by way of electron microscopy. This quantity comprises the most recent thoughts on correlative microscopy displaying that combining imaging modalities presents greater than every one strategy by myself. most significantly it comprises the basic protocols, together with suggestions, tips and photographs so you might repeat those interesting innovations on your personal lab. With state-of-the-art fabric, this finished assortment is meant to steer researchers for years to come.

  • Covers sections on version platforms and practical reports, imaging-based ways and rising studies
  • Chapters are written through specialists within the field
  • Cutting-edge material
  • Second of 2 volumes devoted to Correlative mild and Electron microscopy (CLEM)

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2% GA in 1Â PBS buffer; no osmium or uranyl acetate postfixation. 3 List of embedding resins or mediums and their effects on structure and fluorescence Embedding medium Fluorescent protein (FP) Lowicryl HM20 Nonpolar acrylic resin Low-temperature infiltration Low-temperature UV polymerization Hydrophobic Lowicryl K4M Polar, acrylic resin Low-temperature infiltration Low-temperature UV polymerization Hydrophilic LR White Acrylic resin High-temperature or low-temperature polymerization UV polymerization optional Hydrophilic EGFP; mCherry Publication Notable features/ issues Kukulski et al.

To prevent clustering, sonicate the solution immediately prior to use. 2 Fluorescence microscopy 1. Mount the grid, section side up, on a glass slide in 30 mL of nonhardening mounting medium with coverslip (we use Vectashield® Vector Laboratories, Peterborough, UK). 2. , 10 Â) in bright field, noting orientation of grid on a sheet of paper. Drawing a diagram or printing a large picture of your grid from the manufacturer’s Web site upon which you can sketch locations will help relocate ROIs later in TEM.

The cells on the coverslip were then processed using a modified Tokuyasu cryo-embedding protocol, followed by cryo-sectioning. The resulting 70-nm-thick serial sections were imaged via TEM and compared with confocal images to ensure subregions were relocated within the fluorescence z-stack of the same cell. Testing the utility of live-cell, EM correlated imaging, van Rijnsoever et al. (2008) were able to track and distinguish two differently labeled endosomes in live imaging; one endosome contained either dextran or transferrin-Alexa-568, while another carried both mGFP and either a dextran- or transferrin-bound Alexa568.

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