Leica Science Lab - Tag : High Pressure Freezing https://www.leica-microsystems.com//science-lab/tag/?tx_leicaacademy_pi4%5Baction%5D=show&tx_leicaacademy_pi4%5Bcontroller%5D=Tag&tx_leicaacademy_pi4%5Btag%5D=583&cHash=7bfc3671fc73fb780480703cfba41d78 Article tagged with High Pressure Freezing en-US https://www.leica-microsystems.com/44778 EM Sample Preparation High-pressure freezing: Revealing functional mechanisms of synaptic transmission Learn more about applying optogenetic stimulation in the EM ICE and how this technology has the potential to reveal structural and functional mechanisms of synaptic transmission. Get a detailed introduction of how optogenetic stimulation is applied to intact networks in acute brains slices and organotypic brain slice cultures from mice. https://www.leica-microsystems.com/high-pressure-freezing-revealing-functional-mechanisms-of-synaptic-transmission/ Mon, 14 Dec 2020 08:16:00 +0000 https://www.leica-microsystems.com/26877 EM Sample Preparation Neuroscience Ultrastructural Preservation and Improved Visualization of Membranes in Primary Bovine Chromaffin Cells Intracellular dynamic events, such as protein trafficking, recycling, and degradation as well as signal transduction, to name a few, can be studied using live-cell imaging techniques. However, conventional light microscopes have limited resolution which is not sufficient to visualize cellular processes that occur at the nanometer scale in their cellular context. Electron microscopy is widely employed to visualize molecular complexes, cellular organelles, tissues and whole organisms at the ultrastructural level. https://www.leica-microsystems.com/science-lab/ultrastructural-preservation-and-improved-visualization-of-membranes-in-primary-bovine-chromaffin-cells/ Mon, 21 Oct 2019 13:45:00 +0000 MSc Gianvito Arpino, MD, PhD Ling-Gang Wu, PhD Christopher Bleck https://www.leica-microsystems.com/25036 EM Sample Preparation Expert Knowledge on High Pressure Freezing and Freeze Fracturing in the Cryo SEM Workflow Get an insight in the working methods of the laboratory and learn about the advantages of Cryo SEM investigation in EM Sample Preparation. Find out how high pressure freezing, freeze fracturing and cryo transfer add to the Cryo SEM workflow and how the Leica portfolio ensures the compatibility between these different steps. https://www.leica-microsystems.com/science-lab/expert-knowledge-on-high-pressure-freezing-and-freeze-fracturing-in-the-cryo-sem-workflow/ Thu, 23 May 2019 22:00:00 +0000 Gisela Höflinger https://www.leica-microsystems.com/25022 EM Sample Preparation Bridging Structure and Dynamics at the Nanoscale through Optogenetics and Electrical Stimulation Nanoscale ultrastructural information is typically obtained by means of static imaging of a fixed and processed specimen. However, this is only a snapshot of one moment within a dynamic system in which structures are constantly changing. Exploring specific time points of a dynamic process is therefore a major challenge. Exploring a process at the nanoscale through optogenetics or electrical field stimulation in combination with timed millisecond precision vitrification is a promising technology to overcome this challenge. In the first part of a series of application notes the practical considerations of stimulation-assisted vitrification are discussed. https://www.leica-microsystems.com/science-lab/bridging-structure-and-dynamics-at-the-nanoscale-through-optogenetics-and-electrical-stimulation/ Mon, 20 May 2019 22:00:00 +0000 Dr. Andres Kaech, PhD Frédéric Leroux https://www.leica-microsystems.com/24707 EM Sample Preparation Cryo-SEM analysis of UV light stimulated sun screen lotion The EM ICE is the only available high-pressure freezer combining light stimulation with fast cryo fixation, a module mostly used for optogenetic approaches in neurobiology studies. However, this approach can be also very interesting for pharmaceutical or cosmetic industries to study the influence of UV light exposure on their target structures. This article shows an example on how UV light exposure changes the ultrastructure of sun screen lotion. https://www.leica-microsystems.com/science-lab/cryo-sem-analysis-of-uv-light-stimulated-sun-screen-lotion/ Wed, 06 Mar 2019 23:00:00 +0000 PhD Dietmar Pum, PhD Saskia Mimietz-Oeckler https://www.leica-microsystems.com/24715 EM Sample Preparation Superior Ultrastructural Preservation and Structural Contrast in Drosophila Tissue Optimal structural preservation of tissue can only be obtained by high pressure freezing. However, preparing the samples in optimal conditions is challenging. This article explains in detail how to dissect, high pressure freeze and freeze substitute Droshophila tissue sample to obtain high structural integrity for subsequent electron microscopic analysis. https://www.leica-microsystems.com/science-lab/superior-ultrastructural-preservation-and-structural-contrast-in-drosophila-tissue/ Sun, 03 Mar 2019 23:00:00 +0000 PhD Zulfeqhar Syed, PhD Christopher Bleck https://www.leica-microsystems.com/24565 EM Sample Preparation Expanding the Limits of Electron Microscopy Sample Preparation Capturing the intricate changes in fine structure or in cell dynamics with conventional cryo solutions can be challenging sometimes. Leica Microsystems has developed a new cryo platform, the Leica EM ICE, to help expand the current limits of cryofixation. Watch this free webinar and learn how the Leica EM ICE combines speed, reliability and flexibility to facilitate research in various scientific fields. https://www.leica-microsystems.com/science-lab/expanding-the-limits-of-electron-microscopy-sample-preparation/ Thu, 06 Dec 2018 23:00:00 +0000 PhD Frédéric Leroux https://www.leica-microsystems.com/19756 EM Sample Preparation Practical Applications of Broad Ion Beam Milling Mechanical polishing can be time consuming and frustrating. It can also introduce unwanted artifacts when preparing cross-sectioned samples for electron backscatter diffraction (EBSD) in the scanning electron microscope (SEM) or light microscope investigation. In contrast, ion beam milling can eliminate undesirable artifacts that will hamper your analysis and interpretation. https://www.leica-microsystems.com/science-lab/practical-applications-of-broad-ion-beam-milling/ Thu, 14 Sep 2017 07:50:00 +0000 https://www.leica-microsystems.com/19755 EM Sample Preparation Revealing Cellular Dynamics with Millisecond Precision What if you can dissect the cellular dynamics with millisecond precision? What if you can unravel the morphological transformation of a neuron millisecond by millisecond using electron microscopy? https://www.leica-microsystems.com/science-lab/revealing-cellular-dynamics-with-millisecond-precision/ Wed, 13 Sep 2017 07:50:00 +0000 PhD Shigeki Watanabe, PhD Frédéric Leroux https://www.leica-microsystems.com/19607 EM Sample Preparation Interview with Dr. Shigeki Watanabe on Research in Synaptic Membrane Dynamics Dr. Shigeki Watanabe, principle investigator of the department of Cell Biology at the Johns Hopkins University School of Medicine in Baltimore, held a workshop in Zürich, Switzerland on methods to study synaptic dynamics with millisecond precision. In collaboration with Dr. Andres Käch from the University of Zurich all workshop attendees enjoyed presentations and hands-on sessions on the EM ICE by Leica Microsystems with Light and Electrical Stimulation, revealing the latest developments in brain research. During this workshop Dr. Bernd Sägmüller from Leica Microsystems had the chance for an interview with Dr. Watanabe. https://www.leica-microsystems.com/science-lab/interview-with-dr-shigeki-watanabe-on-research-in-synaptic-membrane-dynamics/ Thu, 06 Jul 2017 23:00:00 +0000 Dr. Bernd Sägmüller, PhD Shigeki Watanabe https://www.leica-microsystems.com/19176 EM Sample Preparation Drosophila larvae - Sample Preparation for Cryo-SEM Application Note for Leica EM ACE900 - Drosophila larvae were sandwiched between two 3 mm aluminum slit carriers with the 100 μm cavities facing each other and high-pressure frozen with a Leica EM HPM100. No ethanol as synchronization media was used, 1-hexadecene was used as filler. The wholes of the slit carriers were filled with filter tips dipped in 1-hexadecene to keep the carrier sandwich complete after freezing. https://www.leica-microsystems.com/science-lab/drosophila-larvae-sample-preparation-for-cryo-sem/ Mon, 30 Jan 2017 09:50:00 +0000 Dr. Andres Kaech, Prof. Damian Brunner https://www.leica-microsystems.com/19179 EM Sample Preparation Giardia lamblia - Sample Preparation for Cryo-SEM Application Note for Leica EM ACE900 - A 100 mesh copper grid (12 um thickness) was dipped into a concentrated Giardia suspension and sandwiched between two flat 3 mm aluminum specimen carriers with scratched surfaces. Subsequently, the sandwich was transferred to the widened hole of a middle plate (3.1 mm diameter). A 50 um spacer ring was added on top and the specimen immediately frozen with an HPM100 high-pressure freezing machine without using alcohol as synchronization fluid. https://www.leica-microsystems.com/science-lab/giardia-lamblia-sample-preparation-for-cryo-sem/ Mon, 30 Jan 2017 09:35:00 +0000 Dr. Andres Kaech, Joe Paulin Zumthor https://www.leica-microsystems.com/18319 EM Sample Preparation Maple (Acer saccharum) Leaves - High Pressure Freezing and Freeze Substitution for TEM Application Note for Leica EM HPM100 - Leaves were immersed in hexadecene and placed under a gentle (0.3 bar) vacuum for 10 minutes to evacuate the internal air spaces. The leaves were then trimmed to fit the carriers and placed in the 200 μm side of a 6 mm Type A specimen carrier. Free space was filled with additional hexadecene after which a 6 mm Type B specimen carrier was placed on top with the flat side down. https://www.leica-microsystems.com/science-lab/maple-acer-saccharum-leaves-high-pressure-freezing-and-freeze-substitution-for-tem/ Mon, 16 Jan 2017 17:08:00 +0000 Dr. Kim Rensing https://www.leica-microsystems.com/18317 EM Sample Preparation High-Pressure Freezing and Freeze Substitution of Hep-2 Cells Infected with Chlamydia pneumoniae Application Note for Leica EM HPM100 - Hep-2 cells infected with Chlamydia pneumoniae were cultured on carbon-coated 6 mm Sapphire discs. Cells were high-pressure frozen in an EM HPM100 using the 6 mm CLEM middle plate with following setup: Sapphire disc with cells, spacer 200 μm, bare Sapphire disc, 2 spacers 200 μm. Ethanol was used as a synchronization fluid to transfer pressure at room temperature prior to cooling. https://www.leica-microsystems.com/science-lab/high-pressure-freezing-and-freeze-substitution-of-hep-2-cells-infected-with-chlamydia-pneumoniae/ Tue, 20 Dec 2016 11:03:00 +0000 Dr. Andres Kaech https://www.leica-microsystems.com/18008 CLEM Fluorescence Microscopy EM Sample Preparation Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy as a Straightforward Tool to Study Host-Pathogen Interactions Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell. https://www.leica-microsystems.com/science-lab/correlative-cryo-fluorescence-and-cryo-scanning-electron-microscopy-as-a-straightforward-tool-to-study-host-pathogen-interactions/ Tue, 13 Dec 2016 07:58:00 +0000 Martin Strnad https://www.leica-microsystems.com/18312 EM Sample Preparation Commercially Available Hand Creams - Sample Preparation for Cryo-SEM Application Note for Leica EM HPM100 - Hand creams having different water contents were applied into the 100 μm cavities of two 3 mm type A sample carriers which were then closed cream sides inwards. The sample assembly was high pressure frozen with a Leica EM HPM100 and moved to a cooled Leica EM VCT100 loading station. https://www.leica-microsystems.com/science-lab/commercially-available-hand-creams-sample-preparation-for-cryo-sem/ Fri, 18 Nov 2016 17:22:00 +0000 Dr. Kim Rensing https://www.leica-microsystems.com/18940 EM Sample Preparation Visualization of Membrane Dynamics with Millisecond Temporal Resolution Application Note for Leica EM ICE, Leica EM AFS2 - Electrical stimulation of neurons combined with high-pressure freezing allows physiological activation of synaptic activity and precise control over the time frame of the induced synaptic activity. https://www.leica-microsystems.com/science-lab/visualization-of-membrane-dynamics-with-millisecond-temporal-resolution/ Mon, 07 Nov 2016 10:53:00 +0000 PhD Shigeki Watanabe https://www.leica-microsystems.com/18149 EM Sample Preparation Structural Study of C. elegans Application Note for Leica EM ICE, Leica EM AFS2 - Wildtype L4 stage C. elegans (N2 strain) were placed in the 100 μm deep side of Lecithin-coated (see detailed protocol*) type A 3 mm Cu/Au carriers (Leica) with extracellular filler containing 1% (w/v) Agarose type IX and 2% (w/v) Bovine Serum Albumin in bacteria medium (see preparation details**) and sandwiched with the flat side of Lecithin-coated type B 3 mm Cu/Au carriers (Leica). Samples were frozen in a high-pressure freezer (Leica EM ICE). https://www.leica-microsystems.com/science-lab/structural-study-of-c-elegans/ Fri, 16 Sep 2016 08:15:00 +0000 E. G. van Donselaar, Dr. Martin Harterink, Drs. C. E. M. Vocking, Dr. Rob Mesman https://www.leica-microsystems.com/18301 EM Sample Preparation Freeze-Fracture Replication of Pyramidal Cells Application Note for Leica EM HPM100 - Frozen samples (90 μm thick slices frozen by HPM100) were inserted into a double replica table and then fractured into two pieces at –130°C (after insertion of the tissue into BAF 060 the samples should be left in the chamber for 20 min to reach the –130°C). https://www.leica-microsystems.com/science-lab/freeze-fracture-replication-of-pyramidal-cells/ Thu, 08 Sep 2016 16:23:00 +0000 Akos Kulik https://www.leica-microsystems.com/18146 EM Sample Preparation Cultured Rat Hippocampal Neurons Application Note for Leica EM ICE - Rat Hippocampal neurons, cultured on 50 μm thick Aclar (Aclar embedding film, EMS) for 19 days, were frozen in the 100 μm deep side of lecithin coated (detailed protocol Appendix I) type A 3 mm Cu/Au carriers (Leica) and sandwiched with the flat side of lecithin coated type B 3 mm Cu/Au carriers (Leica). No additional filler was used, only cell culture medium with the addition of Hepes buffer pH 7.2 to a final concentration of 25 mM. Samples were frozen in a high-pressure freezer (Leica EM ICE). https://www.leica-microsystems.com/science-lab/cultured-rat-hippocampal-neurons/ Mon, 29 Aug 2016 07:12:00 +0000 E. G. van Donselaar, Dr. Martin Harterink, Drs. C. E. M. Vocking, Dr. W. H. Mueller, Prof. Dr. C. C. Hoogenraad https://www.leica-microsystems.com/18295 EM Sample Preparation Cryo-Electron Microscopy of Vitreous Sections (CEMOVIS) of Yeast Application Note for Leica EM HPM100 - The sections are of yeast frozen with a Leica HPM100 high pressure freezer in the copper tube system, the cell paste was mixed with a pH 6.5 MES/dextran buffer so that a final MES concentration of 50mM and a dextran concentration of 20% was achieved. https://www.leica-microsystems.com/science-lab/cryo-electron-microscopy-of-vitreous-sections-cemovis-of-yeast/ Fri, 12 Aug 2016 14:51:00 +0000 Dr. Jonathan O'Driscoll, Dr. Daniel Kofi Clare, Prof. Helen Saibil https://www.leica-microsystems.com/17987 EM Sample Preparation Immuno - Electron Microscopy of High Pressure Frozen and Freeze Substituted Mouse Heart Application Note for Leica EM AFS2 - Mouse heart tissue from wild-type (WT) and αT -catenin KO (KO) mice was excised, immersed in 20% (w/v) BSA and frozen immediately in a high-pressure freezer Leica EM PACT. Freeze subs­titution was carried out using a Leica EM AFS in dry acetone containing 2% ddH2O and 0.1% glutaraldehyde over a 4-days period. https://www.leica-microsystems.com/science-lab/immuno-electron-microscopy-of-high-pressure-frozen-and-freeze-substituted-mouse-heart/ Wed, 27 Jul 2016 15:35:00 +0000 Riet de Rycke https://www.leica-microsystems.com/17979 EM Sample Preparation Cell Envelope Structure of a Gram-negative Thermotolerant y-Proteobacterium Acidithiobacillus sp., Strain HV2/2 and its Interaction with Pyrite Application Note for Leica EM AFS2 - Cells of the the moderately thermophilic Acidithiobacillus sp.strain HV2/2, were centrifuged at 20,0009xg and then cryo-immobilized by high-pressure freezing on gold carriers. https://www.leica-microsystems.com/science-lab/cell-envelope-structure-of-a-gram-negative-thermotolerant-y-proteobacterium-acidithiobacillus-sp-strain-hv22-and-its-interaction-with-pyrite/ Mon, 25 Jul 2016 10:12:00 +0000 Dr. Andreas Klingl https://www.leica-microsystems.com/18049 EM Sample Preparation Neuroscience Symmetric Synapse - Clathrin Coated Endocytosis Pit in the Postsynaptic Dendrite Application Note for Leica EM ICE - WT hippocampal neurons were plated at a density of 80,000 cells/cm2 on 6 mm sapphire disks for 14 days. Sample were frozen using a high-pressure freezer (Leica EM ICE) under a pressure of 2100bar by mounting it into a sandwich support with extracellular solution containing 15% of Ficoll 400, to assess ice crystal damage. The Cryo-fixation was achieved within milliseconds allowing simultaneous immobilization of all macromolecular components. After freezing, sam­ple was transferred into cryovials containing 1% glutaraldehyde, 1% osmium tetroxide, 1% milliQwater in anhydrous acetone and processed in an automated freeze-substitution device (Leica EM AFS2). https://www.leica-microsystems.com/science-lab/symmetric-synapse-clathrin-coated-endocytosis-pit-in-the-postsynaptic-dendrite/ Wed, 20 Jul 2016 08:56:00 +0000 Dr. Shuwen Chang https://www.leica-microsystems.com/17975 EM Sample Preparation Electron Microscopy of High Pressure Frozen and Freeze Substituted Arabidopsis Thaliana Root Tips Cells Application Note for Leica EM AFS2 - Arabidopsis thaliana roots (mutant PIN1pro:PIN1-GFP;bex5-1) were excised, immersed in 20% (w/v) BSA and frozen immediately in a high-pressure freezer. Freeze substitution was carried out using a Leica EM AFS2. https://www.leica-microsystems.com/science-lab/electron-microscopy-of-high-pressure-frozen-and-freeze-substituted-arabidopsis-thaliana-root-tips-cells/ Fri, 08 Jul 2016 10:02:00 +0000 Riet de Rycke https://www.leica-microsystems.com/17379 EM Sample Preparation High Pressure Freezing with Light Stimulation Sun screen lotion was carefully filled in the 100 μm incision of a 3 mm copper/gold plated flat carrier and covered with 3 mm sapphire disk. The sun screen lotion sample was then high pressure frozen with a Leica EM ICE with and subsequently without light stimulation. The light stimulated samples were exposed to a UV light for 500 milliseconds. https://www.leica-microsystems.com/science-lab/high-pressure-freezing-with-light-stimulation/ Fri, 18 Dec 2015 12:01:00 +0000 PhD Dietmar Pum, Dr. Cveta Tomova, PhD Saskia Mimietz-Oeckler https://www.leica-microsystems.com/15596 EM Sample Preparation Transmission Electron Microscopy Sample Preparation Protocols for the Ultrastructural Study of Cysts of Free-living Protozoa Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. https://www.leica-microsystems.com/science-lab/transmission-electron-microscopy-sample-preparation-protocols-for-the-ultrastructural-study-of-cysts-of-free-living-protozoa/ Thu, 02 Jul 2015 17:25:00 +0000 https://www.leica-microsystems.com/15509 CLEM Correlative In-Resin Super-Resolution and Electron Microscopy Using Standard Fluorescent Proteins We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. https://www.leica-microsystems.com/science-lab/correlative-in-resin-super-resolution-and-electron-microscopy-using-standard-fluorescent-proteins/ Thu, 04 Jun 2015 10:28:00 +0000 https://www.leica-microsystems.com/15106 EM Sample Preparation Fluorescence Microscopy Cryo CLEM – the Combination of Cryo Fluorescence Microscopy with Cryo Electron Microscopy Many biological insights can be obtained by combining the power of Fluorescence Microscopy (FM) with that of Electron Microscopy (EM) to study the same sample – this is called Correlative Light and Electron Microscopy (CLEM). In FM, specific proteins can be labelled and identified, and their dynamics and interactions can be visualized in fixed or living cells. https://www.leica-microsystems.com/science-lab/cryo-clem-the-combination-of-cryo-fluorescence-microscopy-with-cryo-electron-microscopy/ Wed, 18 Mar 2015 08:17:00 +0000 PhD Martin Schorb, PhD John A. G. Briggs https://www.leica-microsystems.com/15081 EM Sample Preparation CLEM New Insights into Cilia and Flagella by Cryo-EM Cilia and flagella were the first organelles to be discovered and have been studied for centuries. However, their essential role in humans and how ciliary defects cause diseases are still not well understood. Cryo-EM has recently shed new light on their inner workings and solved some long-standing mysteries, only to raise new questions on how cilia and flagella function. https://www.leica-microsystems.com/science-lab/new-insights-into-cilia-and-flagella-by-cryo-em/ Mon, 16 Feb 2015 09:25:00 +0000 PhD Thomas Heuser https://www.leica-microsystems.com/14887 EM Sample Preparation Video Tutorials: Filling and Assembling of Different Carriers for High-Pressure Freezing High pressure freezing (HPF) is a cryo-fixation method primarily for biological samples, but also for a variety of non-biological materials. It is a technique that yields optimal preservation in many cell types and tissues or in organic and inorganic composites. Most commonly, the high pressure frozen samples are analyzed further with light or electron microscopy after appropriate processing. https://www.leica-microsystems.com/science-lab/video-tutorials-filling-and-assembling-of-different-carriers-for-high-pressure-freezing/ Fri, 05 Dec 2014 08:45:00 +0000 Dr. Cveta Tomova https://www.leica-microsystems.com/14455 EM Sample Preparation Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. https://www.leica-microsystems.com/science-lab/electron-tomography-of-cryo-immobilized-plant-tissue-a-novel-approach-to-studying-3d-macromolecular-architecture-of-mature-plant-cell-walls-in-situ/ Mon, 17 Nov 2014 17:47:00 +0000 https://www.leica-microsystems.com/14406 EM Sample Preparation Brief Introduction to Freeze Fracture and Etching Freeze fracture describes the technique of breaking a frozen specimen to reveal internal structures. Freeze etching is the sublimation of surface ice under vacuum to reveal details of the fractured face that were originally hidden. A metal/carbon mix enables the sample to be imaged in a SEM (block-face) or TEM (replica). It is used to investigate for instance cell organelles, membranes, layers and emulsions. https://www.leica-microsystems.com/science-lab/brief-introduction-to-freeze-fracture-and-etching/ Wed, 01 Oct 2014 09:27:00 +0000 Gisela Höflinger https://www.leica-microsystems.com/13280 EM Sample Preparation Neuroscience Capturing Cellular Dynamics with Millisecond Temporal Resolution The combination of two powerful techniques: optogenetics and high-pressure freezing now makes it possible to visualize a dynamic cellular activity with temporal resolution of 5 milliseconds. By coupling a flash of light with high-pressure freezing, the process of vesicle recycling at the synapses can now be imaged by electron microscopy. https://www.leica-microsystems.com/science-lab/capturing-cellular-dynamics-with-millisecond-temporal-resolution/ Mon, 12 May 2014 13:16:00 +0000 PhD Shigeki Watanabe, PhD Erik M. Jørgensen https://www.leica-microsystems.com/11782 EM Sample Preparation Freeze Substitution of Trypanosoma brucei Chemical fixation of biological specimens for ultrastructural investigation is a relatively slow and selective process, and therefore a common source of artifacts. Freezing, on the other hand, is an excellent method to physically fix biological specimens in their entirety and without delay; the formation of ice crystals large enough to displace cellular material and destroy structures, would be, however, a major issue. https://www.leica-microsystems.com/science-lab/freeze-substitution-of-trypanosoma-brucei/ Tue, 18 Mar 2014 17:25:00 +0000 Harald Kotisch, Dr. Katy Schmidt, Dr. Jan Leunissen, Dr. Guenter Resch https://www.leica-microsystems.com/11809 EM Sample Preparation Brief Introduction to Freeze Substitution Freeze-substitution is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration. During freeze substitution the “frozen” water is dissolved by an organic solvent, which usually also contains chemical fixatives. https://www.leica-microsystems.com/science-lab/brief-introduction-to-freeze-substitution/ Tue, 04 Feb 2014 09:32:00 +0000 Riet de Rycke, Dr. Andres Kaech https://www.leica-microsystems.com/10734 EM Sample Preparation Thermodynamic Considerations Regarding the LN2 in a High Pressure Freezer Employing liquid nitrogen (LN2) as a coolant in the complex process of high pressure freezing raises certain considerations regarding phase transition not only of the liquid sample to be frozen but also of the element used to freeze the sample. According to the phase diagram of nitrogen, changes in temperature or pressure will alter the state of this element. At ambient pressure nitrogen is liquid at temperatures between –209,86 °C and –195,80 °C. It is a rather fragile equilibrium within limited phase boundaries. https://www.leica-microsystems.com/science-lab/thermodynamic-considerations-regarding-the-ln2-in-a-high-pressure-freezer/ Thu, 10 Oct 2013 21:05:00 +0000 Dr. Paul Wurzinger, Dr. Cveta Tomova https://www.leica-microsystems.com/10447 Laser Microdissection Live-Cell Imaging EM Sample Preparation Confocal Microscopy From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. https://www.leica-microsystems.com/science-lab/from-dynamic-live-cell-imaging-to-3d-ultrastructure-novel-integrated-methods-for-high-pressure-freezing-and-correlative-light-electron-microscopy/ Wed, 28 Aug 2013 11:41:00 +0000 https://www.leica-microsystems.com/10062 EM Sample Preparation Safe High Pressure Freezing of Infectious Microorganisms Especially in core EM-facilities one is confronted with material (microorganisms and cells) which are infectious. It is a must to follow the safety rules according to the National regulations. However even when safe laboratories are available it is very convenient to know that the applied high pressure freezing system is in itself safe. https://www.leica-microsystems.com/science-lab/safe-high-pressure-freezing-of-infectious-microorganisms/ Wed, 24 Jul 2013 14:10:00 +0000 PhD Dimitri Vanhecke, Dr. Benoit Zuber, Dr. Silvio D. Brugger, Daniel Studer https://www.leica-microsystems.com/10281 CLEM EM Sample Preparation Live-Cell Imaging Confocal Microscopy A Precise and Rapid Mapping Protocol for Correlative Light and Electron Microscopy of small invertebrate organisms CLEM (correlative live cell and electronmicroscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). https://www.leica-microsystems.com/science-lab/a-precise-and-rapid-mapping-protocol-for-correlative-light-and-electron-microscopy-of-small-invertebrate-organisms/ Tue, 16 Jul 2013 09:26:00 +0000 https://www.leica-microsystems.com/10060 EM Sample Preparation CLEM Brief Introduction to High-Pressure Freezing Water is the most abundant cellular constituent and therefore important for preserving cellular ultra-structure. Currently the only way to fix cellular constituents without introducing significant structural alterations is by cryo-fixation. There are currently two common methods employed; plunge freezing and high pressure freezing. https://www.leica-microsystems.com/science-lab/brief-introduction-to-high-pressure-freezing/ Wed, 12 Jun 2013 17:59:00 +0000 Dr. Cveta Tomova https://www.leica-microsystems.com/9287 EM Sample Preparation Targeting of Peroxisomal Matrix Proteins in the Diatom Phaeodactylum Tricornutum P. tricornutum cells expressing different types of GFP fusion proteins were harvested via centrifugation at 1,500xg and cryoimmobilized by high-pressure freezing on gold carriers. https://www.leica-microsystems.com/science-lab/targeting-of-peroxisomal-matrix-proteins-in-the-diatom-phaeodactylum-tricornutum/ Sun, 17 Mar 2013 23:00:00 +0000 Dr. Andreas Klingl https://www.leica-microsystems.com/5738 EM Sample Preparation Dry Ultrathin Sectioning Combined With High Pressure Freezing We have used cultured UMR106-01 osteoblastic cells to investigate the process of bone mineralization. UMR106-01 cells as well as primary calvarial bone cells assembly spherical extracellular supramolecular protein-lipid complexes, termed biomineralization foci (BMF), in which the first crystals of hydroxyapatite mineral are deposited (Midura et al., 2004; Wang et al., 2004). A major difference between these culture models is the speed with which mineralization occurs, ranging from 12–16 days after plating for primary osteoblastic cells to 88 h for UMR106-01 cells. https://www.leica-microsystems.com/science-lab/dry-ultrathin-sectioning-combined-with-high-pressure-freezing/ Mon, 10 Dec 2012 23:00:00 +0000 Ph.D. Jeff P. Gorski, M.Sc. Nichole T. Huffman, Thérèse Hillman-Marti, Daniel Studer https://www.leica-microsystems.com/10886 Live-Cell Imaging EM Sample Preparation CLEM Postlipolytic Insulin-dependent Remodeling of Micro Lipid Droplets in Adipocytes Despite the lipolysis–lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. https://www.leica-microsystems.com/science-lab/postlipolytic-insulin-dependent-remodeling-of-micro-lipid-droplets-in-adipocytes/ Tue, 15 May 2012 14:14:00 +0000 https://www.leica-microsystems.com/7970 CLEM EM Sample Preparation CLEM: Combining the Strengths of Light and Electron Microscopy In recent years light microscopy studies have been dominated by live cell imaging while electron microscopy has been used for high-resolution studies. Latterly, there has been increasing interest in combining these techniques. This combination is called Correlative Light Electron Microscopy (CLEM). Due to the high resolution made possible by electron microscopy, artefacts induced during preparation of a sample can, however, also be clearly seen. https://www.leica-microsystems.com/science-lab/clem-combining-the-strengths-of-light-and-electron-microscopy/ Mon, 09 Apr 2007 22:00:00 +0000 Professor Paul Verkade