Dataset Information

Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame.

ABSTRACT: Cryomicroscopy is commonly hampered by frost accumulation, reducing the visual clarity of the specimen. Pulling a vacuum or purging with nitrogen gas can greatly reduce the sample chamber's humidity, but at cryogenic temperatures, even minute concentrations of water vapor can still result in frost deposition. Here, a hygroscopic ice frame was created around the specimen to suppress frost growth during cryomicroscopy. Specifically, fluorescently tagged rat brain vessels were frozen on a silicon nitride window with an ice frame, and the luminescence of the fluorescent tag was improved by a factor of 6 compared to a similar specimen in only a nitrogen purge environment. These findings suggest that the simple implementation of a hygroscopic ice frame surrounding the specimen can substantially improve the visual clarity for cryomicroscopy, beyond that of a vacuum or nitrogen purge system.

SUBMITTER: Lowery AW

PROVIDER: S-EPMC9730467 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame.

Lowery Adam W AW Ambi Ashwin A Miller Lisa M LM Boreyko Jonathan B JB

ACS omega 20221122 48

Cryomicroscopy is commonly hampered by frost accumulation, reducing the visual clarity of the specimen. Pulling a vacuum or purging with nitrogen gas can greatly reduce the sample chamber's humidity, but at cryogenic temperatures, even minute concentrations of water vapor can still result in frost deposition. Here, a hygroscopic ice frame was created around the specimen to suppress frost growth during cryomicroscopy. Specifically, fluorescently tagged rat brain vessels were frozen on a silicon n ...[more]

PMID: 36506191

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Project description:Background and aimsSome frost-tolerant herbaceous plants droop and wilt during frost events and recover turgor and posture on thawing. It has long been known that when plant tissues freeze, extracellular ice forms. Distributions of ice and water in frost-frozen and recovered petioles of Trifolium repens and Escholschzia californica were visualized.MethodsPetioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM.Key resultsWith frost-freezing, parenchyma tissues shrank to approx. one-third of their natural volume with marked cytorrhysis of the cells, and massive blocks of extracellular icicles grew under the epidermis (poppy) or epidermis and subepidermis (clover), leaving these layers intact but widely separated from the parenchyma except at specially structured anchorages overlying vascular bundles. On thawing, the extracellular ice was reabsorbed by the expanding parenchyma, and surface tissues again contacted the internal tissues at weak junctions (termed faults). These movements of water into and from the fault zones occurred repeatedly at each frost/thaw event, and are interpreted to explain the turgor changes that led to wilting and recovery. Ice accumulations at tri-cellular junctions with intercellular spaces distended these spaces into large cylinders, especially large in clover. Xylem vessels of frozen petioles were nearly all free of gas; in thawed petioles up to 20 % of vessels were gas-filled.ConclusionsThe occurrence of faults and anchorages may be expected to be widespread in frost-tolerant herbaceous plants, as a strategy accommodating extracellular ice deposits which prevent intracellular freezing and consequent membrane disruption, as well as preventing gross structural damage to the organs. The developmental processes that lead to this differentiation of separation of sheets of cells firmly cemented at determined regions at their edges, and their physiological consequences, will repay detailed investigation.

Project description:In temperate climates, overwintering buds of trees are often less cold hardy than adjoining stem tissues or evergreen leaves. However, data are scarce regarding the freezing resistance (FR) of buds and the underlying functional frost survival mechanism that in case of supercooling can restrict the geographic distribution. Twigs of 37 temperate woody species were sampled in midwinter 2016 in the Austrian Inn valley. After assessment of FR, infrared-video-thermography and cryo-microscopy were used to study the freezing pattern in and around overwintering vegetative buds. Only in four species, after controlled ice nucleation in the stem (-1.6 ± 0.9°C) ice was observed to immediately invade the bud. These buds tolerated extracellular ice and were the most freezing resistant (-61.8°C mean LT50). In all other species (33), the buds remained supercooled and free of ice, despite a frozen stem. A structural ice barrier prevents ice penetration. Extraorgan ice masses grew in the stem and scales but in 50% of the species between premature supercooled leaves. Two types of supercooled buds were observed: in temporary supercooling buds (14 species) ice spontaneously nucleated at -20.5 ± 4,6°C. This freezing process appeared to be intracellular as it matched the bud killing temperature (-22.8°C mean LT50). This response rendered temporarily supercooled buds as least cold hardy. In 19 species, the buds remained persistently supercooled down to below the killing temperature without indication for the cause of damage. Although having a moderate midwinter FR of -31.6°C (LT50), some species within this group attained a FR similar to ice tolerant buds. The present study represents the first comprehensive overview of frost survival mechanisms of vegetative buds of temperate trees. Except for four species that were ice tolerant, the majority of buds survive in a supercooled state, remaining free of ice. In 50% of species, extraorgan ice masses harmlessly grew between premature supercooled leaves. Despite exposure to the same environmental demand, midwinter FR of buds varied intra-specifically between -17.0 and -90.0°C. Particularly, species, whose buds are killed after temporary supercooling, have a lower maximum FR, which limits their geographic distribution.

Dataset Information

Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame.

Publications

Reducing Frost during Cryoimaging Using a Hygroscopic Ice Frame.

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