By Thomas Mueller-Reichert, Paul Verkade

ISBN-10: 0128010754

ISBN-13: 9780128010754

This new quantity of Methods in telephone Biology seems to be at equipment for examining correlative mild and electron microscopy (CLEM). With CLEM, humans attempt to mix some great benefits of either worlds, i.e. the dynamics details received via gentle microscopy and the ultrastructure as supplied by means of electron microscopy. This quantity comprises the most recent ideas on correlative microscopy displaying that combining imaging modalities offers greater than each one procedure on my own. most significantly it comprises the basic protocols, together with counsel, methods and pictures so that you can repeat those fascinating thoughts on your personal lab. With state-of-the-art fabric, this finished assortment is meant to lead researchers for years to come.

  • Covers sections on version platforms and practical reviews, 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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Additional resources for Correlative light and electron microscopy. 2

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Visualizing live dynamics and ultrastructure of intracellular organelles with preembedding correlative light-electron microscopy. Correlative Light and Electron Microscopy, 111, 21–35. , et al. (2000). Correlative light-electron microscopy reveals the tabular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. Journal of Cell Biology, 148, 45–58. , Foran, D. , & Yang, L. (2012). Robust segmentation of overlapping cells in histopathology specimens using parallel seed detection and repulsive level set.

Section the area with cell(s) of interest. Here, we use a Leica UCT Ultramicrotome and transfer 70-nm-thick (thin sections) or 300-nm-thick (tomography) sections onto Formvar-coated copper slot grids. 19. Grids are stained with uranyl acetate and lead citrate and then imaged with a Tecnai-12 BioTwin TEM (FEI Company, Eindhoven, the Netherlands) for thin sections or a Tecnai F30 TEM (FEI Company) for electron tomography: • Cells originally identified by fluorescence microscopy are located at lower/ medium TEM magnifications (between 300 Â through 3000 Â) by comparing the arrangement of cells adjacent, ACLAR sheet grid marks or etches, and other notable features.

Apply fluorescent fiduciary markers to the section surface. We use Invitrogen Molecular Probes™ Blue FluoSphere® fiducials, 200 nm diameter. It is recommended to quench the Blue FluoSpheres® as the spheres are highly fluorescent in the Blue/UV/Cyan/Green epifluorescence channels and can often obfuscate identification of any fluorescently tagged protein. , 2011). 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).

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