This guide provides information on operating the PV60 self-pressurising vessel with the associated hazards and risks when using liquid nitrogen. It will also emphasize several control measures that one should implement when using liquid nitrogen.

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In carbon thin film applications the quality of carbon film production is key for successfully conducting complex experiments. Commonly, carbon coatings are used in sample preparation for TEM (ambient and cryo) imaging, EDS analysis and replicas.

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Transmission Electron Microscopy (TEM) is a high-resolution imaging technique that
provides information about the structure and morphology of specimens. It uses a high
energy electron beam to penetrate samples and renders images using the transmitted
part of the beam.

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The 2017 Nobel prize in cryo-transmitted Electron Microscopy highlighted the significance of direct observation of the molecular world of the cell. Understanding the structure of biomolecules as well as their changes over cell processes plays an important role in curing disease and drug discovery. Download this application note to understand why we use carbon film in grid preparation.

The increasing amount of applications for nano-fibres, especially those produced in the electrospinning process, creates a need to image them in a way that allows their users to examine not only their alignment and diameter, but also their discrete morphology.

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For low-resolution imaging, a majority of the SEM samples can be sputtered or carbon-coated in the pressure range of 1 x 10−1 to 1 x 10−2 mbar. A higher vacuum lower than 1 x 10−3 mbar base pressure allows the sputtering of oxidizing metals. These metals have a lower grain size ideal for high-resolution imaging. Likewise, lower scattering with the background gases enables high purity, high density, amorphous carbon films.

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Published By

Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

Abstract

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.

Authors

• Miroslava Schaffer
• Benjamin D. Engel
• Tim Laugks
• Julia Mahamid
• Jürgen M. Plitzko
• Wolfgang Baumeister

Contact

• [email protected]

• Published By

  Applied Nanoscience, DOI 10.1007/s13204-016-0520-4

  Abstract

  Abstract Nanoscience offers the potential for great advances in medical technology and therapies in the form of nanomedicine. As such, developing controllable, predictable, and effective, nanoparticle-based therapeutic systems remains a significant challenge. Many polymerbased nanoparticle systems have been reported to date, but few harness materials with accepted biocompatibility. Phosphorylcholine (PC) based biomimetic materials have along history of successful translation into effective commercial medical technologies. This study investigated thesynthesis, characterisation, nanoprecipitation, and in vitro cellular uptake kinetics of PC-based polymeric nanoparticle micelles (PNM) formed by the biocompatible and pH responsive block copolymer poly(2-methacryloyloxyethyl phosphorylcholine)-b-poly(2-(diisopropylamino)ethyl methacrylate) (MPC-DPA). Atom transfer radical polymerisation (ATRP), and gel permeation chromatography (GPC) were used to synthesise and characterise the welldefined MPC100-DPA100 polymer, revealing organic GPC, using evaporative light scatter detection, to be more accurate than aqueous GPC for this application. Subsequent nanoprecipitation investigations utilising photoncorrelation spectroscopy (PCS) revealed PNM size increased with polymer concentration, and conferred Cryostability. PNM diameters ranged from circa 64–69 nm, and increased upon hydrophobic compound loading, circa 65–71 nm, with loading efficiencies of circa 60 % achieved, whilst remaining monodisperse. In vitro studies demonstrated that the PNM were of low cellular toxicity, with colony formation and MTT assays, utilising V79 and 3T3 cells, yielding comparable results. Investigation of the in vitro cellular uptake kinetics revealed rapid, 1 h, cellular uptake of MPC100-DPA100 PNM delivered fluorescent probes, with fluorescence persistence for 48 h. This paper presents the first report of these novel findings, which highlight the potential of the system for nanomedicine application development.

  Keywords:  Nanomedicine  Phosphorylcholine  MPCDPA Micelle  Nanoparticle  Drug delivery

  Authors

  • Jonathan P. Salvage
  • Tia Smith
  • Tao Lu
  • Amendeep Sanghera
  • Guy Standen
  • Yiqing Tang
  • Andrew L Lewis

  Contact

  Jonathan P. Salvage [email protected]

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