reSolution_LNT_No1_en - Leica Microsystems

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EDITORIAL CONTENT Title Panchromatic cathodoluminescence image of cassiterite mineral (SnO 2 ) Image Credits Scott Wight1 , Ed Vicenzi2 , Doug Meier1 , and Kurt Benkstein1 1 National Institute of Standards and Technology 2 Smithsonian Institution Source: Smithsonial National Mineral Collection, Preparation: Cut and polished with the Leica EM TXP Data Collection: FEI Company QuantaTM 200F SEM with Gatan, MonoCL4 Elite System 2 reSOLUTION Dear Readers, It gives me great pleasure to welcome you to the Leica NanoTechnology (LNT) edition of reSolution magazine. As it is our first totally exclusive LNT magazine, we decided to provide information on application techniques for both biology and materials sample preparation. All of the articles were prepared by our customers from around the world and I would like to express our thanks to everyone who provided the high quality articles for this edition. Sharing such experiences helps to disseminate techniques and applications around our EM community. To provide further support for issues regarding sample preparation techniques, later in the year we will launch a Leica EM Sample Preparation Science Laboratory online service where you will be able to find even more information about applications and products specific to your needs. Late last year we launched three new products to enhance sample preparation in your laboratory; a new ion beam slope cutter, the TIC 3X, for materials SEM preparation; a critical point dryer, CPD300 - a prerequisite for good SEM preparation for biological and some materials samples; and an entry level high pressure freezer for freezing samples in tubes, the SPF. You can find more information about these instruments on our website at http://www.leica-microsystems.com/products/electron-microscope-sample-preparation/ This year we have also launched some exciting new products. A new family of coaters, the ACE range, was presented at the EMC meeting in Manchester UK. This new generation of coating systems continues in line with our development philosophy, to automate tedious processes and push forward the boundaries of sample preparation in line with the needs of the scientific community. I hope you enjoy this first LNT reSolution magazine and I look forward to your feedback. Happy Reading! ding! Best Wishes, es, Ian Lamswood wood Marketing Manager BIOLOGY Capturing neuro transmitter receptors 04 and ion channels: High-resolution techniques to localize membrane proteins Biological Electron Microscopy at 07 Durham University Dr. Martin W. Goldberg, Durham University, UK Substitutes for Uranyl Acetate in TEM 09 Thin Section Post-Staining Perusing alternatives for staining applications for TEM thin sections Dry ultrathin sectioning combined with 13 high pressure freezing/freeze-substitution improves retention and visualization of calcium and phosphorus ions prior to nucleation of mineral crystals within osteoblastic cultures INDUSTRY A Word on Cathodoluminescence 17 Atomic Force Microscopy Study of a 19 Stretched Impact Copolymer University of Wollongong Electron 21 Microscopy Centre REGISTRATION 23 IMPRINT 23
Capturing Neurotransmitter Receptors and Ion Channels High-resolution Techniques to Localize Membrane Proteins Daniel Althof1,2 , Akos Kulik1,3 1Department of Anatomy and Cell Biology, Institute of Neuroanatomy 2Spemann Graduate School of Biology and Medicine, University of Freiburg, Germany 3Department of Physiology II, University of Freiburg, Germany Neurotransmitter receptors and ion channels in the central nervous system are localized to synaptic and extrasynaptic membrane compartments of pre- and postsynaptic elements of neurons. The impact of the activation of these proteins on synaptic integration and regulation of transmitter release depends on their precise location relative to synapses, as well as on the density and coupling of molecules in microcompartments of the cells. High-resolution qualitative and quantitative visualization of membranebound receptors and ion channels is, therefore, essential for understanding their roles in cell communication. The ability of the nervous system to learn and respond to the environment refl ects an underlying capability of neurons to dynamically alter the number, type, and strengths of their connections. These connections, called synapses, are highly organized sites of contact between postsynaptic neurons and presynaptic terminals. The specialized synaptic membrane, contains a large variety of molecules such as receptors, ion channels, and associated structural proteins, whose precise subcellular organization facilitates its proper function. A large number of studies have provided evidence that the location of these proteins and their position relative to synapses substantially affects their functional roles. Neurotransmitter receptors, localized to the synaptic membrane of postsynaptic compartments of neurons, are directly exposed to released neurotransmitters. Consequently, they are activated in a transient manner, generating fast postsynaptic responses that are precisely time-locked to the presynaptic action potentials. In contrast, receptors localized to the extrasynaptic plasma membrane, remote from synaptic sites, are activated by spilledover neurotransmitters producing a tonic conductance that is not precisely time-locked to single presynaptic action potentials, but rather refl ects the whole network activity on a slower time scale 1 . Receptors can also be located presynaptically either on the extrasynaptic membrane of axon terminals or over the presynaptic grid where they are activated by neurotransmitters released by the same or by neighbouring boutons. Like neurotransmitter receptors, ion channels are also localized to the somato-dendritic membranes and axon terminals of neurons. Postsynaptic channels are generally playing a role in the integration and plasticity of synaptic inputs, as well as in the control of neuronal excitation by mediating slow inhibitory synaptic responses and contributing to the resting membrane potential. Presynaptic channels that are concentrated either at the presynaptic active zone or localized to the extrasynaptic membrane of boutons are involved in the regulation of neurotransmitter release, thereby playing a role in the presynaptic modulation of neuronal activity. It is, therefore, easy to understand that the same receptor and ion channel could fulfi ll very different functional requirements when targeted to different subcellular compartments of cells 1 . Furthermore, the impact of the activation of membrane proteins on synaptic integration and regulation of transmitter release critically depends on the density and functional coupling of receptors and ion channels in compartments of the target neurons, as well as on their location relative to excitatory and inhibitory synaptic sites. Thus, the question arises of how the precise subcellular location of these molecules can be determined at high resolution. For this purpose the following advanced high-resolution immunocytochemical methods have been widely used: (i) preembedding immunogold, (ii) postembedding immunogold, and (iii) sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) techniques. (i) In case of the preembedding immunogold method, an 0.8 nm or a 1.4 nm gold particle is coupled to the secondary antibodies Fig. 1 BIOLOGY Akos Kulik Daniel Althof NANOTECHNOLOGY 3
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