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专业: 半导体晶体与薄膜材料

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标题：Epiphytic cyanophytes Xenococcus  kerneri and Chamaesiphon minutus on the freshwater red alga Paralemanea catenata (Rhodophyta)
[ Biologia February 2006, Volume 61, Issue 1, pp 11-13 Pavel Kučera Bohuslav Uher Ondřej Komárek]
摘要：Epiphytes of Paralemanea catenata (Kützing) Vis & Sheath (Rhodophyta) were studied on material from the River Dyje in National Park Podyjí. Longitudinal sections of the thalli were made with help of Cryo Cut Microtome techniques. The epiphytic cyanophytes Xenococcus kerneri Hansgirg and Chamaesiphon minutus (Rostafinski) Lemmermannwere recognized on the cortex of the thalli. Our observations of the determined species are discussed.
Key words:
epiphytic Cyanophytes- Xenococcus- Chamaesiphon- Paralemanea- Czech Republic

标题：Chapter 6 – 3D HDO-CLEM: Cellular Compartment Analysis by Correlative Light-Electron Microscopy on Cryosection
[Katia Cortese∗,, 1, Giuseppe Vicidomini§,, 1, Maria Cristina Gagliani∗, Patrizia Boccacci∗, Alberto Diaspro∗,, †,, ‡,, §, Carlo Tacchetti∗,, †, ║ ]

Fundamental to obtaining a depth-understanding of the function and structure of cells is the ability to study and correlate their molecular topography with the ultrastructural morphology, for example, to visualize the position of a given protein relative to a given cell compartment and its morphology. Standard fluorescence light microscopy (FLM) relies on simple sample preparations, and localizes proteins in living or fixed cells with a resolution in the range of few hundred nanometers, allowing large field of view. However, FLM is unable to visualize the unlabeled cellular context. On the other hand, electron microscopy (EM) techniques reveal protein topology with the resolution in a range of a few tens of nanometer, retains the cellular context, but can only be applied on a limited field of view. Therefore, both approaches present shortcomings, in terms of field of view, statistical output, resolution, sample preparation, and context analysis, that can likely complement each other. To bridge the gap between FLM imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In a nutshell, CLEM enables one to investigate the same exact region of interest utilizing the two microscope platforms, and thereby virtually combine their capabilities.
In this chapter, we describe a protocol based on immunolabeling of Tokuyasu cryosections that allows correlation of LM and EM images with excellent preservation of cellular ultrastructure. We will refer to this method as high-data-output CLEM (HDO-CLEM).The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision of transmission EM in a CLEM hybrid morphometric analysis.
We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and set the basis for a hybrid light/EM morphometry approach.
Keywords
Confocal microscopy; 共焦显微镜
Correlative microscopy; 
Cryosection; 
Electron microscopy;
Immunofluorescence; 
Immunogold; 
Morphometry形态测量学

标题：A new approach to improve the quality of ultrathin cryo-sections; its use for immunogold EM and correlative electron cryo-tomography
[Corresponding author at: Division of Cell Biology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek, Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.Copyright © 2011 Elsevier Inc. All rights reserved.]
Abstract
Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing; this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their ‘close to native’ cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample.
Keywords
Cryo-sections; 
Cryo-ultramicrotomy; 
Immunogold labeling; 
Vitrification; 
Correlative electron cryo-tomography

标题：Chapter 11 – Immuno Correlative Light and Electron Microscopy on Tokuyasu Cryosections
[Viola M.J. Oorschot*,  Tamar E. Sztal†,  Robert J. Bryson-Richardson†,  Georg Ramm]
Abstract
Finding a rare structure by electron microscopy is the equivalent of finding a “needle in a haystack.” Correlative light- and immunoelectron microscopy (CLEM) on Tokuyasu cryosections is a sophisticated technique to address this challenge. Hereby, fluorescently labeled structures of interest are identified in an overview image by light microscopy and subsequently traced in electron microscopy. While the direct transfer and imaging of the same sections from optical to electron microscopy enables straightforward correlation, the sample preparation is crucial and technically demanding. We provide a detailed guide outlining the critical steps for sample embedding, cryosectioning, immunolabeling, and imaging. In the example provided, we use CLEM to trace aggregates formed in a zebrafish myopathy model expressing enhanced green fluorescent protein (eGFP) tagged actin. In our case, only a few muscle fibers express eGFP-actin with a subset of fibers containing aggregates. By fluorescence microscopy, we are able to identify the aggregates in the zebrafish tissue, and we subsequently, use immunoelectron microscopy to image the same structures at high resolution. The CLEM method described here using Tokuyasu cryosections can be applied to a large range of samples including small organisms, tissue samples, and cells.
Keywords
Immuno-EM; 
CLEM; 
Tokuyasu; 
Zebrafish; 
GFP

   标题：Electron Tomography of Immunolabeled Cryosections
  [Mark S. Ladinsky*,   Kathryn E. Howell ]
Publisher Summary
Electron tomography (ET) provides an informative view of cellular fine structure in three dimensions (3D). Immunolabeling provides the localization of specific macromolecules to a structure of interest. Combining the two techniques by means of preparing tomographic reconstructions of immunolabeled samples results in a powerful method for localizing macromolecules within the 3D context of a cell. This chapter focuses mostly on the Golgi complex in cells or tissues that are immobilized by rapid freezing, then fixed by freeze-substitution, and subsequently embedded in plastic. This methodology has helped in deciphering the complex structure of the Golgi with a high degree of confidence. A modified version of the method for immunolabeling cryosections is valuable for studies of the Golgi complex and associated vesicles, tubules, and organelles. Although this modified method is somewhat limited by its use of chemical fixation, it has proven most efficient for the localization of molecules in membranous compartments such as the Golgi complex and endoplasmic reticulum (ER).

标题：Strategies to improve the antigenicity, ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue
[York-Dieter Stierhofa, , ,  Farid El Kasmib ]
  Abstract
  Immunolabelling of (ultra)thin thawed cryosections according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for cells and tissues. However, chemical fixation at ambient temperature, a prerequisite of this technique, can cause problems for samples, like plant tissue, because cell walls, hydrophobic surfaces and intercellular air slow down diffusion of fixative molecules into the sample. We show that a hybrid technique, based on a combination of cryofixation/freeze-substitution and Tokuyasu cryosection immunolabelling, circumvents the disadvantages associated with chemical fixation and results in an improved ultrastructure and antigenicity preservation of Tokuyasu cryosections used for light and electron microscopic immunolabelling (as shown for Myc- or mRFP-tagged proteins, KNOLLE and carbohydrate epitopes). In combination with the most sensitive particulate marker systems, like 1-nm gold or quantum dot markers, we were able to obtain a differentiated labelling pattern which allows a more detailed evaluation of plant Golgi, trans-Golgi network and multivesicular body/prevacuolar compartment markers (COPI-specific γCOP, the ADP-ribosylation factor GTPase ARF1, ARA7/RabF2b and the vacuolar sorting receptor VSR). We also discuss possibilities to improve membrane contrast, e.g., of transport vesicles like COPI, COPII and clathrin-coated vesicles, and of compartments of endosomal trafficking like the trans-Golgi network.
Keywords
Arabidopsis; 
Freeze-substitution; 
High-pressure freezing; 
Immunofluorescence labelling;
Immunogold labelling; 
Quantum dot; 
trans-Golgi network; 
Tokuyasu cryosection;
Trafficking; 
VAMP727
标题：Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)
Cryo-scanning
[Keita Endoh ;  Jun Kasuga ; Keita Arakawa; Toshiaki Ito; Seizo Fujikawa ]
  Abstract
The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.
Keywords
Dormant bud; 休眠芽
Larch (Larix kaempferi); 日本落叶松
Extraorgan freezing; 
Cryo-scanning electron microscope (Cryo-SEM); 
Deep supercooling

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