High Vacuum Requirements Research Articles

Evaluation of green laser source additive manufacturing technology for accelerator applications with ultra-high vacuum requirements

Additive Manufacturing (AM) offers different benefits such as efficient material usage, reduced production time and design freedom. Moreover, with continuous technological developments, AM expands in versatility and different material usage capabilities. Recently new energy sources have been developed for AM – green wavelength lasers, which provide better energy absorption for pure copper. Due to high thermal and electrical conductivity of copper, this novel AM technology is highly promising for various industries, particularly, there is a huge interest to use it for accelerator applications. In particular, these AM produced accelerator components should reach the associated Ultra High Vacuum (UHV) requirements. In this study, vacuum membranes of pure copper were produced by AM using a green laser source, in different thicknesses and built angles. Furthermore, a vacuum membrane helium leak tightness test was performed at room temperature by using a high-sensitivity mass spectrometer. Comparison of these test results was performed with previously established results. Through this study, novel knowledge and initial results are provided for green laser source AM technology usage for applications for UHV accelerator components.

Procedure qualification and integration of the continuous external Rogowski diagnostics to the superconducting magnets of ITER tokamak

Continuous External Rogowski (CER) are the primary plasma current diagnostics for the ITER tokamak, as well as the only diagnostics built into the ITER Toroidal Field Coils (TF coils). The integration of CER diagnostics to the TF coils faced numerous technical challenges, such as: the integrated components have to withstand the maximum pressure foreseen during ITER TF coil resin impregnation (350 kPa), comply with the high vacuum requirements that the helium leak rate shall be <2.7 × 10−9 Pa-m3/s, and survive at cryogenic temperature. The CER diagnostics integration campaign turned out to be a marked success, thanks to a comprehensive pre-qualification program and well-coordinated collaborations among the participating parties. All four sets of CER diagnostics have so far been integrated to the respective TF coils. As a pioneering attempt of building plasma diagnostics into reactor-scale superconducting magnets, the potential implications of this venture to the diagnostics development for DEMO and future fusion reactors are discussed. The gained experiences and lessons learned from this venture are summarized at last.

Compact and modular system architecture for a nano-resonator-mass spectrometer.

Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution.

The restriction of calcium influx in metaphase and post-metaphase stages of cell division revealed by imaging secondary ion mass spectrometry (SIMS).

A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK1 cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to 44 Ca stable isotope in the extracellular buffer for 10min. The 44 Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 (40 Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of 44 Ca in 10min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the 44 Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.

Advancing Plasmon-Induced Selectivity in Chemical Transformations with Optically Coupled Transmission Electron Microscopy

Nanoparticle photocatalysts are essential to processes ranging from chemical production and water purification to air filtration and surgical instrument sterilization. Photochemical reactions are generally mediated by the illumination of metallic and/or semiconducting nanomaterials, which provide the necessary optical absorption, electronic band structure, and surface faceting to drive molecular reactions. However, with reaction efficiency and selectivity dictated by atomic and molecular interactions, imaging and controlling photochemistry at the atomic scale are necessary to both understand reaction mechanisms and to improve nanomaterials for next-generation catalysts. Here, we describe how advances in plasmonics, combined with advances in electron microscopy, particularly optically coupled transmission electron microscopy (OTEM), can be used to image and control light-induced chemical transformations at the nanoscale. We focus on our group's research investigating the interaction between hydrogen gas and Pd nanoparticles, which presents an important model system for understanding both hydrogenation catalysis and hydrogen storage. The studies described in this Account primarily rely on an environmental transmission electron microscope, a tool capable of circumventing traditional TEM's high-vacuum requirements, outfitted with optical sources and detectors to couple light into and out of the microscope. First, we describe the H2 loading kinetics of individual Pd nanoparticles. When confined to sizes of less than ∼100 nm, single-crystalline Pd nanoparticles exhibit coherent phase transformations between the hydrogen-poor α-phase and hydrogen-rich β-phase, as revealed through monitoring the bulk plasmon resonance with electron energy loss spectroscopy. Next, we describe how contrast imaging techniques, such as phase contrast STEM and displaced-aperture dark field, can be employed as real-time techniques to image phase transformations with 100 ms temporal resolution. Studies of multiply twinned Pd nanoparticles and high aspect ratio Pd nanorods demonstrate that internal strain and grain boundaries can lead to partial hydrogenation within individual nanoparticles. Finally, we describe how OTEM can be used to locally probe nanoparticle dynamics under optical excitation and in reactive chemical environments. Under illumination, multicomponent plasmonic photocatalysts consisting of a gold nanoparticle "antenna" and a Pd "reactor" show clear α-phase nucleation in regions close to electromagnetic "hot spots" when near plasmonic antennas. Importantly, these hot spots need not correspond to the traditionally active, energetically preferred sites of catalytic nanoparticles. Nonthermal effects imparted by plasmonic nanoparticles, including electromagnetic field enhancement and plasmon-derived hot carriers, are crucial to explaining the site selectivity observed in PdHx phase transformations under illumination. This Account demonstrates how light can contribute to selective chemical phenomena in plasmonic heterostructures, en route to sustainable, solar-driven chemical production.

New status of the infrared beamlines at SSRF

There are two infrared beamlines at the Shanghai synchrotron radiation facility (SSRF)—BL01B and BL06B. BL01B was the first infrared beamline of the National Facility for Protein Science in Shanghai at SSRF, which is dedicated to synchrotron infrared microspectroscopy. It utilizes bending magnet radiation and edge radiation as light sources. Diffraction-limited spatial resolution is reached in the infrared microspectroscopy experiment. BL01B has been in operation for approximately five years since it opened in January 2015. In the past few years, many meaningful results have been published by user groups from various disciplines, such as biomacromolecule materials and pharmaceutical, environmental, and biomedical sciences. In addition, a new infrared beamline station BL06B is under construction, which is optimized for the mid-infrared and far-infrared band. BL06B is equipped with a vacuum-type Fourier transform infrared spectrometer, infrared microscope, custom long-working-distance infrared microscope, infrared scanning near-field optical microscope, and mid-infrared Mueller ellipsometer. The purpose is to serve experiments with high vacuum requirements and spatial resolution experiments, as well as those that are in situ and time-sensitive, such as high-pressure and atomic force microscopy infrared experiments. The station can be used for research in biomaterials, pharmacy, geophysics, nanotechnology, and semiconductor materials.

Dynamic imaging of Au-nanoparticles via scanning electron microscopy in a graphene wet cell

High resolution nanoscale imaging in liquid environments is crucial for studying molecular interactions in biological and chemical systems. In particular, electron microscopy is the gold-standard tool for nanoscale imaging, but its high-vacuum requirements make application to in-liquid samples extremely challenging. Here we present a new graphene based wet cell device where high resolution scanning electron microscope (SEM) and energy dispersive x-rays (EDX) analysis can be performed directly inside a liquid environment. Graphene is an ideal membrane material as its high transparancy, conductivity and mechanical strength can support the high vacuum and grounding requirements of a SEM while enabling maximal resolution and signal. In particular, we obtain high resolution ( nm) SEM video images of nanoparticles undergoing Brownian motion inside the graphene wet cell and EDX analysis of nanoparticle composition in the liquid enviornment. Our obtained resolution surpasses current conventional silicon nitride devices imaged in both a SEM and transmission electron microscope under much higher electron doses.

MEMS Carbon Nanotubes Field Emission Pressure Sensor With Simplified Design: Performance and Field Emission Properties Study

The pressure sensor application gained recently substantial interest in many fields of basic and applied research and applications. In this paper, microelectromechanical system (MEMS)-based pressure sensor contains nanostructured electrode consisting of carbon nanotube (CNT) array. CNTs are directly grown on such electrode by plasma-enhanced chemical vapor deposition method using microwave plasma torch at atmospheric pressure. This growth method enables us to use a simple electrode structure without need of buffer layer and time-consuming lithography process. Combination of CNTs field emission and MEMS membrane mechanical properties make possible to enhance sensitivity of the sensor. Field emission properties of CNTs are measured by newly developed system enabling us precise measurement of expecting properties, such as dependence on diaphragm (upper electrode) distance, applied voltage, and stability of the sensor. Measured values are compared with a numerical modeling of the membrane system in Coventor Ware software by finite-element method. We also suggest encapsulating the sensor using glass frit bonding because such method is more suitable for high vacuum requirements of the field emission operation.

Development and Integration Testing of a Power Coupler for a 3.9-GHz Superconducting Multicell Cavity Resonator

A coaxial power coupler for a superconducting multicell cavity resonator at 3.9 GHz has been developed. The cavity is intended to be employed as an accelerator to provide enhanced electron beam quality in a free-electron laser. Due to cryogenic high-vacuum and high-power requirements, special provisions for two windows and two bellows were implemented. A simulation tool was employed to optimize the coupler structure for low reflection of incident power and dissipation while restraining the field at critical locations to prevent material breakdown. The procedures for testing the coupler on its own and integrated with the superconducting cavity are described, and the measurement results are presented. The coupler-cavity assembly was tested to exceed the requirement of 9.3-kW input power and axial field intensity of 14.5 MV/m in the cavity. Coupler return and insertion losses were estimated to be 21 and 0.2 dB, respectively.

Properties of CuIn1-xGaxSe2 films prepared by the rapid thermal annealing of spray-deposited CuIn1-xGaxS2 and Se

ABSTRACTMany reported CuIn1-xGaxSe2 (CIGS) thin films for high-efficiency solar cells have been prepared via a two-stage process that consists of a high-vacuum film deposition step followed by selenization with excess H2Se gas or Se vapor. Removing toxic gas and high-vacuum requirements from this process would greatly simplify it and make it less hazardous. We report the formation of CuIn1-xGaxSe2 (x = 0, 0.25, 0.50, 0.75, 1.0) thin films achieved by rapid thermal annealing of spray-deposited CuIn1-xGaxS2 and Se in the absence of an additional selenium source. To prepare the Se layer, commercial Se powder was dissolved by refluxing in ethylenediamine/2,2-dimethylimidizolidine. After cooling to room temperature, this mixture was combined with 2-propanol and the resulting colloidal Se suspension was sprayed by airbrush onto a heated glass substrate. The resulting film was coated with nanocrystalline CuIn1-xGaxS2 via spray deposition of a toluene-based “nanoink” suspension. The two-layer sample was annealed at 550 oC in an argon atmosphere for 60 minutes to form the final CIGS product. Scanning electron microscopy images reveal that film grains are 200-300 nm in diameter and comparable to sizes of the reactant CuIn1-xGaxS2 nanoparticles. XRD patterns are consistent with the chalcopyrite unit cell and calculated lattice parameters and A1 phonon frequencies change nearly linearly between those for CuInSe2 and CuGaSe2.

Measuring fundamental properties in operating solid oxide electrochemical cells by using in situ X-ray photoelectron spectroscopy

Photoelectron spectroscopic measurements have the potential to provide detailed mechanistic insight by resolving chemical states, electrochemically active regions and local potentials or potential losses in operating solid oxide electrochemical cells (SOCs), such as fuel cells. However, high-vacuum requirements have limited X-ray photoelectron spectroscopy (XPS) analysis of electrochemical cells to ex situ investigations. Using a combination of ambient-pressure XPS and CeO(2-x)/YSZ/Pt single-chamber cells, we carry out in situ spectroscopy to probe oxidation states of all exposed surfaces in operational SOCs at 750 °C in 1 mbar reactant gases H(2) and H(2)O. Kinetic energy shifts of core-level photoelectron spectra provide a direct measure of the local surface potentials and a basis for calculating local overpotentials across exposed interfaces. The mixed ionic/electronic conducting CeO(2-x) electrodes undergo Ce(3+)/Ce(4+) oxidation-reduction changes with applied bias. The simultaneous measurements of local surface Ce oxidation states and electric potentials reveal the active ceria regions during H(2) electro-oxidation and H(2)O electrolysis. The active regions extend ~150 μm from the current collectors and are not limited by the three-phase-boundary interfaces associated with other SOC materials. The persistence of the Ce(3+)/Ce(4+) shifts in the ~150 μm active region suggests that the surface reaction kinetics and lateral electron transport on the thin ceria electrodes are co-limiting processes.

Preparation of plasma-polymerized para-xylene as an alternative to parylene coating for biomedical surface modification

Abstract Parylene (or poly-para-xylene) coating is known to be a transparent, uniform and effective barrier. There are however some drawbacks, including an expensive starting material, high thermal energy consumption for monomer generation, high vacuum requirements and a low growth rate. In this study, low-cost para-xylene was used as the starting material to form plasma-polymerized films. A pulsed-dc plasma power supply was used and the deposits were examined for their microstructure, mechanical properties and fibroblast cytotoxicity. Experimental results reveal that the proposed coatings exhibit an amorphous structure with a deposition rate widely ranging from 50 to 480 nm/h, depending on the pulse frequency (ωp) of the input power and monomer flow rate (fp). At high fp and low ωp, the plasma-polymerized para-xylene (PPX) films exhibit alkane and alkene features, indicating a more organic character. In contrast, the films obtained at low fp and high ωp present inorganic features. For its mechanical properties, a pencil hardness of 7H–8H in the coated specimen is higher than that of the conventional parylene coating, and the film adhesion graded at 4B determined using the cross-cut test is similar to that of the conventional parylene coating. The water contact angle of PPX films in our study was measured and ranged from 60° to 85°, which is more hydrophilic than the conventional parylene coating. Cell cultures on PPX deposited specimens present a higher cell count than the parylene deposited film. The PPX films deposited at high ωp and low fp exhibit a lower water contact angle (hydrophilic), which accounts for its better cell compatibility. These quantitative indications imply that the PPX is a possible alternative to the conventional parylene coating for biomedical device surface modification.

Thermal structural analysis of SST-1 vacuum vessel and cryostat assembly using ANSYS

Steady state super-conducting tokamak-1 (SST-1) is a medium sized tokamak, which has been designed to produce a ‘D’ shaped double null divertor plasma and operate in quasi steady state (1000 s). SST-1 vacuum system comprises of plasma chamber (vacuum vessel, interconnecting rings, baking and cooling channels), and cryostat all made of SS 304L material designed to meet ultra high vacuum requirements for plasma generation and confinement. Prior to plasma shot and operation the vessel assembly is baked to 250/150 °C from room temperature and discharge cleaned to remove impurities/trapped gases from wall surfaces. Due to baking the non-uniform temperature pattern on the vessel assembly coupled with atmospheric pressure loading and self-weight give rise to high thermal-structural stresses, which needs to be analyzed in detail. In addition the vessel assembly being a thin shell vessel structure needs to be checked for critical buckling load caused by atmospheric and baking thermal loads. Considering symmetry of SST-1, 1/16th of the geometry is modeled for finite element (FE) analysis using ANSYS for different loading scenarios, e.g. self-weight, pressure loading considering normal operating conditions, and off-normal loads coupled with baking of vacuum vessel from room temperature 250 °C to 150 °C, buckling and modal analysis for future dynamic analysis. The paper will discuss details about SST-1 vacuum system/cryostat, solid and FE model of SST-1, different loading scenarios, material details and the stress codes used. We will also present the thermal structural results of FE analysis using ANSYS for various load cases being investigated and our observations under different loading conditions.

Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2 +) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 1 5N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

UHV compatible Al to SS joining through electro magnetic forming technique

Fusion welding, which is the most versatile and widely used method of metallurgical bonding, can be troublesome in certain applications like dissimilar metal joining for Ultra High Vacuum requirements. Welding of Stainless Steel to Aluminum by conventional technique is very difficult due to thermal & metallurgical incompatibility. Consequently a number of non-conventional specialized joining techniques were developed. Joining of SS to Al by Electro Magnetic Forming (EMF) method is an effort in this direction. The paper discusses in detail the joining process, joint design and the results achieved.

A New High Resolution Field Emission SEM with Variable Pressure Capabilities

Abstract To examine non conductive samples in their natural state (i.e. without significant sample preparation) at high resolution in the SEM the technique of low voltage field emission scanning electron microscopy (LVFESEM) is used. Due to the limitation in accelerating voltage (U&amp;lt;1kV) this technique is limited in respect of chemical analysis. Furthermore it is not possible to examine humid and outgassing samples in high vacuum. in recent years the application of variable pressure scanning electron microscopes (VPSEM) became an important technique in materials science as well as in life science. Due to the capability of maintaining a high chamber pressure humid, outgassing and non-conductive samples, can be examined in their natural state without significant sample modification or preparation. Especially compound materials with different electron yields can be imaged without any charging effects (Fig. 2), [2]. This paper describes a high resolution field emission electron microscope, that combines low voltage and variable pressure capabilities. The high pressure capabilities of the instrument are realized by eliminating the high vacuum requirements of SEM in the microscope chamber. This is done by separating the vacuum environment in the chamber from the ultra high vacuum environment in the gun area.

Portable double‐focusing mass‐spectrometer system for field gas monitoring

AbstractTraditional mass‐spectrometry (MS) instruments have been confined to laboratory usage because of their large size, power and high‐vacuum requirements, which make them unsuitable for use in most field environments. Developments in mass spectrometers and small vacuum pump systems have now made possible the development of transportable mass‐spectrometer instruments. A portable MS device based on a miniature double‐focusing (ExB) sector‐field mass spectrometer has been developed for in situ gas analysis and monitoring applications. The device (acronym Portable‐CDFMS) uses a 1.1‐Tesla NdFeB permanent magnet, a lithographically defined electric sector, a molecular‐flow frit inlet, a spiral filament in a closed ion source, and a miniature channeltron detector. The high vacuum is provided with the use of a very compact hybrid turbomolecular and diaphragm pump combination. This MS system weighs 18 lb., is able to detect atmospheric species at the part‐per‐million (ppm) level, and has a mass range up to 200 amu, which makes this miniature mass spectrometer well suited for field applications. © 2001 John Wiley &amp; Sons, Inc. Field Analyt Chem Technol 5: 156–167, 2001

Photoresponse of a miniaturized ultrabroad-band low-noise metal-film bolometer detector array

A miniaturized low-noise metal-film bolometer detector array capable of recording radiation spanning a wide spectral range is presented. The bolometers consist of structured gold layers coated with a black nickel layer to reduce the reflectivity in the long-wavelength range. Investigation of the device`s photoresponse showed sufficient sensitivity at wavelengths lambda =4 mm to 6 AA. The detector unit meets high-vacuum requirements, is bakeable up to 300 degrees C, can withstand high nuclear radiation doses, is insensitive to thermal and electromagnetic interferences and can be operated in high magnetic fields. The cooling time of the bolometer is determined by its thermal properties, being about 70 ms in air. The device has meanwhile been successfully used in a few fusion plasma experiments.

Liquid sample introduction for matrix-assisted laser desorption ionization

One of the most important tools in analytical chemistry is liquid chromatography with mass spectrometric detection (LC/MS). The development of interfaces between liquid separation and mass spectrometry is a difficult problem because the large volume flow of liquid solvent in the LC effluent is incompatible with the high vacuum requirements of a mass spectrometer. In this paper initial results are presented for a new liquid introduction method for a mass spectrometer based on the application of matrix assisted laser desorption ionization (MALDI) to aerosols. Details of the method will be presented in future publications. Aerosols are formed using a continuous pneumatic aerosol generator from an acidified methanol solution containing the matrix and analyte. The solvent is removed by passing the aerosol through a heated tube to form a collimated beam of dried particles. Ions are formed by irradiating the aerosol with pulsed UV radiation in the source region of a linear TOF MS. The advantages of the aerosol MALDI TOF method are (1) liquid flow capacity of 0.5 mL/min, (2) rapid acquisition of the entire mass spectrum, and (3) ionization of large peptides and proteins. In this paper, the aerosol MALDI mass spectra of gramicidin S and lysozyme (chicken eggmore » white) are presented. Flow injection mass spectra of gramicidin S are presented to demonstrate the feasibility of coupling aerosol MALDI with HPLC. 22 refs., 6 figs.« less

The structure of grease via electron microscopy and image analysis

AbstractMany of the insolubles that are essential to the structure of lubricating greases are too small to be usefully imaged in the optical microscope. The higher resolution of the electron microscope is necessary to examine the thickener and additive morphology that contribute so much to the performance of modern greases. However, the high vacuum requirements of these instruments mean that the oil component of grease should not be introduced into the microscope at ambient temperatures. Techniques have been developed to remove the oil prior to examination, or to reduce its vapour pressure to a negligible level. A number of such techniques which have proved to be of value for both the scanning (SEM) and transmission (TEM) microscopes are described and illustrated. The distribution of the thickener fibres can now be measured and analysed by an image analysis procedure from the images of freeze‐fracture TEM replicas.