Webinar on Demand: High Pressure Freezing With Leica EM ICE - The Tool For Understanding Dynamic Processes
In this webinar, we will introduce the features and applications of the Leica EM ICE high pressure freezer with light stimulation, emphasizing the physics and technical solutions behind the user interface. We will discuss parameters such as dwell time, pressure and temperature correlation, how they are measured and what the displayed data means. We will also explain how light stimulation is synchronized with the freezing process to give you millisecond precision in your experiments.
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.
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).
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).
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.
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.
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.
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.
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.
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.
Capturing Neurotransmitter Receptors and Ion Channels
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.
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.
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.