High-pressure Samples Research Articles

Falcon Probe: A High-Pressure and Robust Sampling Interface for Coupling Lossless Liquid Chromatography Injection with In Situ Nanoliter-Scale Sample Pretreatment.

With the increasing demand for trace sample analysis, injecting trace samples into liquid chromatography-mass spectrometry (LC-MS) systems with minimal loss has become a major challenge. Herein, we describe an in situ LC-MS analytical probe, the Falcon probe, which integrates multiple functions of high-pressure sample injection without sample loss, high-efficiency LC separation, and electrospray. The main body of the Falcon probe is made of stainless steel and fabricated by the computer numerical control (CNC) technique, which has ultrahigh mechanical strength. By coupling a nanoliter-scale droplet reactor made of polyether ether ketone (PEEK) material, the Falcon probe-based LC-MS system was capable of operating at mobile-phase pressures up to 800 bar, which is comparable to those of conventional ultraperformance liquid chromatography (UPLC) systems. Using the probe pressing microamount in situ (PPMI) injection approach, the Falcon probe-based LC-MS system showed high separation efficiency and good repeatability with relative standard deviations (RSDs) of retention time and peak area of 1.8% and 9.9%, respectively, in peptide mixture analysis (n = 6). We applied this system to the analysis of a trace amount of 200 pg of HeLa protein digest and successfully identified an average of 766 protein groups (n = 5). By combining in situ sample pretreatment at the nanoliter range, we further applied the present system in single-cell proteomic analysis, and 241 protein groups were identified in single 293 cells, which preliminarily demonstrated its potential in the analysis of trace amounts of samples with complex compositions.

Sourcing and reducing sample environment background in low-temperature high-pressure neutron scattering experiments

Neutron scattering in high-pressure environments offers unique opportunities to measure the physical properties of matter but faces challenges due to low signal intensity and high background noise. In this study, Monte Carlo simulations were employed to identify background sources in low-temperature and high-pressure sample environments at the SINQ instrument CAMEA. Simulation results suggest that the piston pressure cell (PC) contributes significantly to the background noise in neutron scattering experiments. To counter this background, a compact radial neutron collimator was developed to reduce background in the sample environment, and its performance was evaluated experimentally. Measurements show that the collimator reduces the background generated by the PC effectively, especially for low q. Noise from the collimator was observed, and strategies to address this issue were discussed.

High-pressure X-ray photon correlation spectroscopy at fourth-generation synchrotron sources.

A new experimental setup combining X-ray photon correlation spectroscopy (XPCS) in the hard X-ray regime and a high-pressure sample environment has been developed to monitor the pressure dependence of the internal motion of complex systems down to the atomic scale in the multi-gigapascal range, from room temperature to 600 K. The high flux of coherent high-energy X-rays at fourth-generation synchrotron sources solves the problems caused by the absorption of diamond anvil cells used to generate high pressure, enabling the measurement of the intermediate scattering function over six orders of magnitude in time, from 10-3 s to 103 s. The constraints posed by the high-pressure generation such as the preservation of X-ray coherence, as well as the sample, pressure and temperature stability, are discussed, and the feasibility of high-pressure XPCS is demonstrated through results obtained on metallic glasses.

Submillisecond in situ X-ray diffraction measurement system with changing temperature and pressure using diamond anvil cells at BL10XU/SPring-8.

Recently, there has been a high demand for elucidating kinetics and visualizing reaction processes under extreme dynamic conditions, such as chemical reactions under meteorite impact conditions, structural changes under nonequilibrium conditions, and in situ observations of dynamic changes. To accelerate material science studies and Earth science fields under dynamic conditions, a submillisecond in situ X-ray diffraction measurement system has been developed using a diamond anvil cell to observe reaction processes under rapidly changing pressure and temperature conditions replicating extreme dynamic conditions. The development and measurements were performed at the high-pressure beamline BL10XU/SPring-8 by synchronizing a high-speed hybrid pixel array detector, laser heating and temperature measurement system, and gas-pressure control system that enables remote and rapid pressure changes using the diamond anvil cell. The synchronized system enabled momentary heating and rapid cooling experiments up to 5000 K via laser heating as well as the visualization of structural changes in high-pressure samples under extreme dynamic conditions during high-speed pressure changes.

Influence of high-pressure heat treatment on magnetocaloric effects and magnetic phase transition in single crystal Gd3Ga5O12

The magnetic properties and magnetic phase transition critical behavior of Gd3Ga5O12 single crystals subjected to high-pressure heat treatment were investigated. The results show that high-pressure heat treatment reduces the Curie temperature and magnetization of the sample. Under a magnetic field change of 5 T, the maximum isothermal magnetic entropy of the sample is approximately 19.73 J/(kg·K). High-pressure heat treatment increases the phase transition temperature range and leads to an increase in the magnetic refrigeration power. Both Gd3Ga5O12 single crystals and the high-pressure heat-treated sample undergo a second-order phase transition. The critical behavior of the samples aligns with the mean field model acquired via critical model fitting. This indicates that the samples exhibit long–range exchange interactions in the system near the Curie temperature. Thus, this material can be used as a magnetic refrigerant for low-temperature applications.

A versatile pressure-cell design for studying ultrafast molecular-dynamics in supercritical fluids using coherent multi-pulse x-ray scattering.

Supercritical fluids (SCFs) can be found in a variety of environmental and industrial processes. They exhibit an anomalous thermodynamic behavior, which originates from their fluctuating heterogeneous micro-structure. Characterizing the dynamics of these fluids at high temperature and high pressure with nanometer spatial and picosecond temporal resolution has been very challenging. The advent of hard x-ray free electron lasers has enabled the development of novel multi-pulse ultrafast x-ray scattering techniques, such as x-ray photon correlation spectroscopy (XPCS) and x-ray pump x-ray probe (XPXP). These techniques offer new opportunities for resolving the ultrafast microscopic behavior in SCFs at unprecedented spatiotemporal resolution, unraveling the dynamics of their micro-structure. However, harnessing these capabilities requires a bespoke high-pressure and high-temperature sample system that is optimized to maximize signal intensity and address instrument-specific challenges, such as drift in beamline components, x-ray scattering background, and multi-x-ray-beam overlap. We present a pressure cell compatible with a wide range of SCFs with built-in optical access for XPCS and XPXP and discuss critical aspects of the pressure cell design, with a particular focus on the design optimization for XPCS.

High-pressure vapor-liquid equilibrium measurements of methane + water mixtures by nuclear magnetic resonance spectroscopy

The vapor-liquid equilibrium (VLE) of methane + water mixtures has been studied with nuclear magnetic resonance (NMR) spectroscopy. This work had two primary goals. The first goal was to develop methods that broaden the utility of NMR spectroscopy for VLE measurements. In this regard, we report a method by which the liquid-phase and vapor-phase compositions are measured in separate experiments by adjusting the height of the liquid phase in the sample tube. We also report a method for hastening phase equilibration by adding glass beads to the sample and repeatedly inverting the sample tube. The second goal of this work was to collect VLE data on a challenging mixture with real-world importance. Mixtures of methane + water are a useful test case because of their challenging characteristics, including the widely differing vapor pressures of the two components. One use for accurate VLE data on methane + water mixtures is to better predict the formation of harmful liquid phases in natural gas pipelines. Herein we utilize 1H NMR spectroscopy to measure the VLE of methane + water mixtures at temperatures of 299.73, 307.98, and 323.25 K, and pressures ranging from 0.69 MPa to 13.89 MPa. Experiments were carried out with a 600 MHz spectrometer. Mixtures were prepared and equilibrated in a high-pressure zirconia sample tube with an integrated needle valve. NMR-based VLE measurements on the liquid phase are in good agreement with available literature data and with Henry's Law predictions at low pressures. However, the commonly used GERG-2008 model for natural gas systems deviates dramatically from the experimental data for the liquid phase. NMR-based VLE measurements on the vapor-phase resulted in measured water concentrations that are systematically lower than available literature data and models. This systematic offset is likely caused by peak overlap in the NMR spectra.

Dynamic optical spectroscopy and pyrometry of static targets under optical and x-ray laser heating at the European XFEL

Experiments accessing extreme conditions at x-ray free electron lasers (XFELs) involve rapidly evolving conditions of temperature. Here, we report time-resolved, direct measurements of temperature using spectral streaked optical pyrometry of x-ray and optical laser-heated states at the High Energy Density instrument of the European XFEL. This collection of typical experiments, coupled with numerical models, outlines the reliability, precision, and meaning of time dependent temperature measurements using optical emission at XFEL sources. Dynamic temperatures above 1500 K are measured continuously from spectrally- and temporally-resolved thermal emission at 450–850 nm, with time resolution down to 10–100 ns for 1–200 μs streak camera windows, using single shot and integrated modes. Targets include zero-pressure foils free-standing in air and in vacuo, and high-pressure samples compressed in diamond anvil cell multi-layer targets. Radiation sources used are 20-fs hard x-ray laser pulses at 17.8 keV, in single pulses or 2.26 MHz pulse trains of up to 30 pulses, and 250-ns infrared laser single pulses. A range of further possibilities for optical measurements of visible light in x-ray laser experiments using streak optical spectroscopy are also explored, including for the study of x-ray induced optical fluorescence, which often appears as background in thermal radiation measurements. We establish several scenarios where combined emissions from multiple sources are observed and discuss their interpretation. Challenges posed by using x-ray lasers as non-invasive probes of the sample state are addressed.

Use of finite element analysis for the thermal gradient determination of a neutron transparent high-pressure sample environment for neutron tomography

ABSTRACT A 3D Finite Element Analysis model was developed to describe the temperature distribution inside a novel neutron transparent high-pressure sample assembly. The validity of the model was established by conducting experimental validation. The Finite Element Analysis model was utilised to evaluate the sample assembly’s temperature gradients and optimise its geometry and components. The results indicate that the discrepancy between the temperature recorded in the laboratory using thermocouples and the temperature calculated by the Finite Element Analysis model was only 4% under the assumption that the thermocouple was positioned in the middle of the sample assembly. The models further demonstrate that the Finite Element Analysis approach is a valuable tool for optimising the sample assembly by considering the impact of different materials and variations in the shape of its components.

Fabrication of pea protein gels with modulated rheological properties using high pressure processing

High pressure processing (HPP) as a non-thermal food preservation technology has recently received growing interest in protein processing. In the present study, the impact of high-pressure level, treatment time, protein concentration, and sample pH on the pea protein gel microstructure and strength were systematically investigated, as well as the protein conformation study, and the gel rheological and dissolution tests. A minimum of 10% and 15% (w/v) protein concentration was required for self-standing pea protein gel formation under 600 MPa and 300 MPa, respectively, at ambient temperature. The pH played an important role in affecting the gel microstructures and properties using HPP treatment. Particulate gels with compact structures and high mechanical strength up to 130 kPa were obtained at pH 5 mainly stabilized by hydrophobic interactions. Whereas fine-stranded gels mainly stabilized by hydrogen bonding with good elasticity and high water-holding capacity were formed at pH 7. The pH, HPP pressure level, and duration have been modulated to prepare a series of pea protein gels to mimic the texture of jelly, pudding, and tofu products. The tunable capacity using HPP makes pea protein a potential gelling ingredient in a wide range of food applications as a substitute for animal or soy protein. Furthermore, the gels treated with pH-shifting followed by HPP showed high freeze-thaw stability with a minimum syneresis level of 15% after two freeze-thaw cycles, demonstrating their potential to be used in frozen food products. Considering the advantages such as improved nutritional quality and extended shelf life compared to thermal technologies, HPP could be a promising non-thermal technique for the fabrication of pea protein gels with tunable rheological and mechanical properties to meet consumer needs for diversified texture and sensory quality.

Heat capacity of microgram oxide samples by fast scanning calorimetry.

Quantitative scanning calorimetry on microgram-sized samples opens a broad, new range of opportunities for studying the thermodynamic properties of quantity-limited materials, including those produced under extreme conditions or found as rare accessory minerals in nature. We calibrated the Mettler Toledo Flash DSC 2+ calorimeter to obtain quantitative heat capacities in the range 200-350 °C, using samples weighing between 2 and 11.5 μg. Our technique is applied to a new set of oxide materials to which it has never been used before, without the need for melting, glass transitions, or phase transformations. Heat capacity data were obtained for silica in the high pressure stishovite (rutile) structure, dense post-stishovite glass, standard fused quartz, and for TiO2 rutile. These heat capacities agree within 5%-15% with the literature values reported for rutile, stishovite, and fused SiO2 glass. The heat capacity of post-stishovite glass, made by heating stishovite to 1000 °C, is a newly reported value. After accurate calibrations, measured heat capacities were then used to calculate masses for samples in the microgram range, a substantial improvement over measurement in conventional microbalances, which have uncertainties approaching 50%-100% for such small samples. Since the typical uncertainty of heat capacities measured on 10-100 mg samples in conventional differential scanning calorimetry is typically 7% (1%-5% with careful work), flash differential scanning calorimetry, using samples a factor of 1000 smaller, increases the uncertainty of heat capacity measurements by a factor of <3, opening the door for meaningful measurements on ultra-small, high-pressure samples and other quantity-limited materials.

Quality Determination of a High-Pressure Processed Avocado Puree-Based Smoothie Beverage

The aim was to study the quality stability of a high-pressure (HP) processed avocado puree-based smoothie beverage and to determine its shelf life. To achieve this mathematical description of HP process parameters (pressure, temperature, and pH conditions) on polyphenoloxidase (PPO) inactivation of avocado-puree (base of the smoothie beverage), use of the appropriate kinetic models was undertaken. Inactivation rate constants were obtained for combinations of constant pressure (600, 700, 750 MPa) and temperature (25, 35, 45 °C) for pH values 4 and 5. According to the Eyring and Arrhenius equations, activation volumes and activation energies, respectively, representing pressure and temperature dependence of the inactivation rate constant, were calculated for all temperatures and pressures studied. The combined use of HP led to PPO inactivation (&lt;10% remaining PPO activity). An increase in the temperature at pressure 600 or 750 MPa caused an increase in PPO inactivation (4.5 and 9.0%, respectively). The ultimate goal was to produce a HP processed avocado puree-based smoothie beverage (containing acid whey and other ingredients) with superior quality and increased shelf life (under refrigeration). The blended ingredients were HP processed in PET packages (600 MPa-25 °C-10 min, 600 MPa-35 °C-10 min, 750 MPa-25 °C-5 min, 750 MPa-35 °C-5 min) based on PPO inactivation as well as industrial practices. Non-processed as well as thermally (TM) processed (90 °C-5 min) samples were used as control samples. No significant differences were found in sensorial attributes between non-processed and HP samples, although the aroma and acceptability scores decreased significantly for thermally pasteurized smoothies. Based on the data obtained, 600 MPa-25/35 °C-10 min are sufficient to obtain safe smoothies (of pH 5 approximately) (up to 6 months) whose organoleptic properties are equally as acceptable to consumers as freshly made smoothies.

Effect of perceptive-auditory training on the classification of speech hypernasality.

To analyze the effect of auditory-perceptual training by inexperienced speech-language pathologists in the classification of hypernasality in individuals with cleft lip and palate and compare their classification of hypernasality individually, with the gold standard evaluation, before and after this training. Three inexperienced speech-language pathologists used a four-point scale to assess 24 high-pressure speech samples from individuals with cleft lip and palate, before and after auditory-perceptual training. The speech samples corresponded to six samples of each degree of hypernasality. The speech-language pathologists received auditory-perceptual training during the assessments. They had access to anchor samples and immediate feedback of correct answers regarding the degree of hypernasality in training. There was no significant difference in the overall percentage of correct answers when comparing before and after the auditory-perceptual training. There was a significant association and agreement of the three evaluators with a gold standard evaluation after training, with an increase in agreement for a single evaluator for absent and mild degrees of hypernasality. The dichotomous analysis of the data showed an increase in the Kappa Index of Agreement of this evaluator. Although there was an increase in the Index of Agreement between evaluators for absent, mild, and severe hypernasality, this increase did not reach statistical significance. The auditory-perceptual training provided did not result in a significant improvement in the hypernasality classification for the inexperienced speech-language pathologists, even though the individual data analysis showed that the training favored one of the evaluators. Further studies involving gradual and more extensive auditory-perceptual training may favor the classification of hypernasality by inexperienced SLPs.

An integrated strategy of secondary metabolomics and glycomics to investigate multi-component variations in wine-processing of medicinal herbs and functional foods: A case study on Fructus Corni

Fructus Corni (FC), as a promising Chinese medicinal herb, has aroused considerable interest. Generally, FC needs to be processed according to the limited standard policy in China before clinical application, while the investigations on the specific processing methods (such as wine steaming or high-pressure wine steaming) are unclear. A comprehensive metabolomics strategy based on integrated non-targeted metabolomics and targeted glycomics in this paper was implemented to investigate the influences of the different processing technologies such as steaming, wine steaming, high-pressure steaming, high-pressure wine steaming, wine immersion, and wine stir-frying on FC, respectively. UHPLC-Q-TOF-MS/MS was employed for identifying and distinguishing the secondary metabolites. A total of 85 components were identified in all groups. The results of PCA score plots showed that the crude and processed samples had a complete separation, and wine steamed and high-pressure wine steamed samples could be a category, indicating that the two processed products had a similar quality. Multiple chromatography including HPLC (C18)-PDA, HPLC (NH2)-ELSD, and HPGPC-ELSD was used for determining the molecular weight distributions, the monosaccharide compositions of polysaccharides, and the contents of free monosaccharides and oligosaccharides. The results indicated that the content and composition of saccharides were different in crude and different processed FC. The polysaccharides were composed of fucose, arabinose, galactose, glucose, galacturonic acid, mannose and rhamnose, and the free monosaccharides were composed of fucose, arabinose, galactose, glucose, mannose, rhamnose and fructose in all FC samples. The PCA score plots of the glycomics indicated that the crude and high-pressure wine steamed FC could be a category, showing that the two groups had similar chemical compositions. Ultimately, the simulation processing experiments indicated that the transformation of morroniside, polysaccharides, oligosaccharides, fructose, and glucose to 5-HMF through the reactions of dehydration and deglycosylation was the potential mechanism of enhancing the effects by processing. Conclusionly, the saccharides should be investigated as thoroughly as the secondary metabolites, and the high-pressure wine steamed FC could be an alternative to wine steamed FC.

Structural changes induced by pasteurisation and/or high-pressure treatment of skim caprine milk

The effects of pasteurisation, high-pressure (HP), and a combination of pasteurisation and high-pressure (PHP) on the physicochemical properties and protein structure of caprine skim milk was investigated. Samples treated by PHP generally had a higher pH, whey protein denaturation, surface hydrophobicity, and intrinsic fluorescence than those treated only with heat or pressure. In contrast, the size of skim milk casein micelles decreased significantly with an increase in pressure level and time; however, the effect was less marked when heat and pressure treatments were combined. For the PHP and HP samples, as the level and time of pressure increased, the α-helix and β-turn content reduced, whereas β-sheet and random coil were induced. Thus, PHP treatment could be used as a good alternative technology in the dairy industry to promote the functional properties of skim caprine milk.

Preservation of high-pressure volatiles in nanostructured diamond capsules.

High pressure induces dramatic changes and novel phenomena in condensed volatiles1,2 that are usually not preserved after recovery from pressure vessels. Here we report a process that pressurizes volatiles into nanopores of type 1 glassy carbon precursors, converts glassy carbon into nanocrystalline diamond by heating and synthesizes free-standing nanostructured diamond capsules (NDCs) capable of permanently preserving volatiles at high pressures, even after release back to ambient conditions for various vacuum-based diagnostic probes including electron microscopy. As a demonstration, we perform a comprehensive study of a high-pressure argon sample preserved in NDCs. Synchrotron X-ray diffraction and high-resolution transmission electron microscopy show nanometre-sized argon crystals at around 22.0 gigapascals embedded in nanocrystalline diamond, energy-dispersive X‑ray spectroscopy provides quantitative compositional analysis and electron energy-loss spectroscopy details the chemical bonding nature of high-pressure argon. The preserved pressure of the argon sample inside NDCs can be tuned by controlling NDC synthesis pressure. To test the general applicability of the NDC process, we show that high-pressure neon can also be trapped in NDCs and that type 2 glassy carbon can be used as the precursor container material. Further experiments on other volatiles and carbon allotropes open the possibility of bringing high-pressure explorations on a par with mainstream condensed-matter investigations and applications.

Global halogen flux of subducting oceanic crust

In order to constrain the geochemical cycling of halogens (F, Cl, Br, I) during subduction of altered ocean crust (AOC), this study compares bulk halogen concentrations from seven seafloor AOC drill cores (n=21) and exhumed eclogites (n=22) from three paleo-subduction settings (Raspas Complex, Ecuador; Zambezi Belt, Zambia; Cabo Ortegal, Spain). As ocean crust undergoes hydrothermal alteration, basalts and gabbros preferentially incorporate different halogens. Fluorine is predominantly hosted in basalts (averaging 155 μg/g); Cl and Br are enriched in gabbros (averaging 241 μg/g and 225 ng/g, respectively). During prograde metamorphism of AOC, F behaves compatibly and is decoupled from the heavy halogens (Cl, Br, I). Eclogite samples retain F in concentrations (30–160 μg/g) similar to seafloor values (20–190 μg/g). Chlorine and Br are strongly coupled and effectively mobilized during prograde metamorphism, with high-pressure (HP) samples containing between 5–15 μg/g Cl and 17–69 ng/g Br. Elevated F/Cl, Br/Cl, and I/Cl in eclogites relative to MORB and AOC ratios support the decoupling of halogens during metamorphism as Cl is most efficiently expelled from the slab; the mobility of halogens in subducting AOC is as follows: Cl≈Br>I≫F. In conjunction with published values of halogen abundances in seafloor serpentinites, marine sediments, and HP metaserpentinites and metasediments, the results of this study are used to estimate global halogen fluxes. Mass balance estimates indicate that up to 2% of the initial F, 50% of the initial Cl, 93% of the initial Br, and 97% of initial I entering subduction zones is lost between the trench and eclogite facies. Subducted AOC represents a substantial halogen reservoir to arc magma depths and may represent the most significant carrier of halogens to the deep mantle.

Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation.

Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their disease-specific biomolecular cargoes and importance in cell-cell communications. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of nanoscale EVs (≈50-200nm) from endogenous proteins in biological samples. Herein, a unique microfluidic platform is reported for EV-protein fractionation based on the principle of size exclusion chromatography (SEC). Using a novel rapid (≈20min) replica molding technique, a fritless microfluidic SEC device (μSEC) is fabricated using thiol-ene polymer (UV glue NOA81, Young's modulus ≈1GPa) for high pressure (up to 6bar) sample processing. Controlled on-chip nanoliter sample plug injection (600 nL) using a modified T-junction injector is first demonstrated with rapid flow switching response time (<1.5 s). Device performance is validated using fluorescent nanoparticles (50nm), albumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, EVs are directly isolated from undiluted human platelet-poor plasma using μSEC and show distinct elution profiles between EVs and proteins based on nanoparticle particle analysis (NTA), Western blot and flow cytometry analysis. Overall, the optically transparent μSEC can be readily automated and integrated with EV detection assays for EVs manufacturing and clinical diagnostics.

Supercritical ion exchange: A new method to synthesize copper exchanged zeolites

A new technique termed Supercritical Ion Exchange (SCIE) was developed and used to synthesize Cu-mordenite (Cu-MORS). The ion exchange takes place between the Cu complex (Copper(II)trifluoroacetylacetonate) dissolved in supercritical CO2 (scCO2) and the extraframework protons in mordenite zeolite without requiring an aqueous phase. The occurrence of the ion exchange reaction was demonstrated by using 1H NMR analysis of the high-pressure fluid phase samples and by visual inspection of the fluid phase color change during the synthesis. SCIE resulted in selective ion-exchange inferred by the equilibrium isotherm. The prepared catalysts were used for the stepwise direct methane to methanol (sDMTM) process and results showed that methanol productivity increased linearly with increasing Cu loading up to a certain Cu wt%. Cu-MORS displayed 16% higher methanol productivity as compared to Cu-MORA (prepared by aqueous ion exchange) with the same Cu loading. The results demonstrated the importance of site selective ion-exchange for zeolite catalysis.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.