Zooplankton boom and ice amphipod bust below melting sea ice in the Amundsen Gulf, Arctic Canada

Early summer in the Arctic with extensive ice melt and break-up represents a dramatic change for sympagic–pelagic fauna below seasonal sea ice. As part of the International Polar Year-Circumpolar Flaw Lead system study (IPY-CFL), this investigation quantified zooplankton in the meltwater layer below landfast ice and remaining ice fauna below melting ice during June (2008) in Franklin Bay and Darnley Bay, Amundsen Gulf, Canada. The ice was in a state of advanced melt, with fully developed melt ponds. Intense melting resulted in a 0.3- to 0.5-m-thick meltwater layer below the ice, with a strong halocline to the Arctic water below. Zooplankton under the ice, in and below the meltwater layer, was sampled by SCUBA divers. Dense concentrations (max. 1,400 ind. m⁻³) of Calanus glacialis were associated with the meltwater layer, with dominant copepodid stages CIV and CV and high abundance of nauplii. Less abundant species included Pseudocalanus spp., Oithona similis and C. hyperboreus. The copepods were likely feeding on phytoplankton (0.5–2.3 mg Chl-a m⁻³) in the meltwater layer. Ice amphipods were present at low abundance (<10 ind. m⁻²) and wet biomass (<0.2 g m⁻²). Onisimus glacialis and Apherusa glacialis made up 64 and 51% of the total ice faunal abundance in Darnley Bay and Franklin Bay, respectively. During early summer, the autochthonous ice fauna becomes gradually replaced by allochthonous zooplankton, with an abundance boom near the meltwater layer. The ice amphipod bust occurs during late stages of melting and break-up, when their sympagic habitat is diminished then lost.     

Late Quaternary stratigraphy and sedimentology of Orphan Basin: Implications for meltwater dispersal in the southern Labrador Sea

Orphan Basin is a deep-water basin on the continental margin off Newfoundland, which throughout the late Quaternary received proglacial sediment from local ice that crossed the continental shelf. Sediment from more distant sources was transported southward in the Labrador Current as proglacial plumes and in icebergs. Five sedimentary facies related to glacial processes are distinguished in cores recovered from Orphan Basin: hemipelagic sediment, nepheloid-layer deposits (layered mud), beds rich in ice-rafted detritus (IRD), sand and mud turbidites, and glaciogenic debris-flow deposits. IRD-rich beds correspond to periods of intensified iceberg calving, and layered mud, turbidites, and glaciogenic debris-flow deposits with glacial meltwater discharge.

In the Late Wisconsinan, eight periods of meltwater discharge and iceberg calving from the Newfoundland ice sheet are interpreted from the sediment facies in Orphan Basin. These discharges coincide with the terminations of the colder periods of the D–O cycles recorded in Greenland ice cores. The oldest minor meltwater event (27.5–28.5 cal ka) corresponds to the first Late Wisconsinan ice advance across the Grand Banks and NE Newfoundland Shelf. The following three meltwater discharges (23–23.5, 23.8–24.5, and 25–27 cal ka) deposited sand turbidites and glaciogenic debris-flow deposits seaward of Trinity Trough, which was occupied by an ice stream at this time, and mud turbidites in the southern part of the basin derived from a mid-shelf ice margin on the Grand Banks. Four periods of meltwater discharge occurred during the deglaciation and are centered at 15, 18.5, 19.75, and 20.75 cal ka. The youngest is correlated to Heinrich event 1. In the literature, the 18.5 and 20.75 cal ka events have been recorded in multiple glacial settings in the North Atlantic, and therefore, are interpreted as large-scale events of meltwater discharge and iceberg calving, but in Orphan Basin the 19.75 cal ka event is also of similar scale.     

Distinct bacterial communities exist beneath a high Arctic polythermal glacier

Bacterial communities reside in basal ice, sediment, and meltwater in the supra-, sub-, and proglacial environments of John Evans Glacier, Nunavut, Canada. We examined whether the subglacial bacterial community shares common members with the pro- and supraglacial communities, and by inference, whether it could be derived from communities in either of these environments (e.g., by ice overriding proglacial sediments or by in-wash of surface meltwaters). Terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes amplified from these environments revealed that the subglacial water, basal ice, and sediment communities were distinct from those detected in supraglacial meltwater and proglacial sediments, with 60 of 142 unique terminal restriction fragments (T-RFs) detected exclusively in subglacial samples and only 8 T-RFs detected in all three environments. Supraglacial waters shared some T-RFs with subglacial water and ice, likely reflecting the seasonal flow of surface meltwater into the subglacial drainage system, whereas supraglacial and proglacial communities shared the fewest T-RFs. Thus, the subglacial community at John Evans Glacier appears to be predominantly autochthonous rather than allochthonous, and it may be adapted to subglacial conditions. Chemical analysis of water and melted ice also revealed differences between the supraglacial and proglacial environments, particularly regarding electrical conductivity and nitrate, sulfate, and dissolved organic carbon concentrations. Whereas the potential exists for common bacterial types to be broadly distributed throughout the glacial system, we have observed distinct bacterial communities in physically and chemically different glacial environments.     

Distinct Bacterial Communities Exist beneath a High Arctic Polythermal Glacier

Bacterial communities reside in basal ice, sediment, and meltwater in the supra-, sub-, and proglacial environments of John Evans Glacier, Nunavut, Canada. We examined whether the subglacial bacterial community shares common members with the pro- and supraglacial communities, and by inference, whether it could be derived from communities in either of these environments (e.g., by ice overriding proglacial sediments or by in-wash of surface meltwaters). Terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes amplified from these environments revealed that the subglacial water, basal ice, and sediment communities were distinct from those detected in supraglacial meltwater and proglacial sediments, with 60 of 142 unique terminal restriction fragments (T-RFs) detected exclusively in subglacial samples and only 8 T-RFs detected in all three environments. Supraglacial waters shared some T-RFs with subglacial water and ice, likely reflecting the seasonal flow of surface meltwater into the subglacial drainage system, whereas supraglacial and proglacial communities shared the fewest T-RFs. Thus, the subglacial community at John Evans Glacier appears to be predominantly autochthonous rather than allochthonous, and it may be adapted to subglacial conditions. Chemical analysis of water and melted ice also revealed differences between the supraglacial and proglacial environments, particularly regarding electrical conductivity and nitrate, sulfate, and dissolved organic carbon concentrations. Whereas the potential exists for common bacterial types to be broadly distributed throughout the glacial system, we have observed distinct bacterial communities in physically and chemically different glacial environments.     

Cryoconite pans on Snowball Earth: supraglacial oases for Cryogenian eukaryotes?

Geochemical, paleomagnetic, and geochronological data increasingly support the Snowball Earth hypothesis for Cryogenian glaciations. Yet, the fossil record reveals no clear‐cut evolutionary bottleneck. Climate models and the modern cryobiosphere offer insights on this paradox. Recent modeling implies that Snowball continents never lacked ice‐free areas. Wind‐blown dust from these areas plus volcanic ash were trapped by snow on ice sheets and sea ice. At a Snowball onset, sea ice was too thin to flow and ablative ice was too cold for dust retention. After a few millenia, sea ice reached 100 s of meters in thickness and began to flow as a ‘sea glacier’ toward an equatorial ablation zone. At first, dust advected to the ablative surface was recycled by winds, but as the surface warmed with rising CO₂, dust aka cryoconite began to accumulate. As a sea glacier has no terminus, cryoconite saturated the surface. It absorbed solar radiation, supported cyanobacterial growth, and sank to an equilibrium depth forming holes and decameter‐scale pans of meltwater. As meltwater production rose, drainages developed, connecting pans to moulins, where meltwater was flushed into the subglacial ocean. Flushing cleansed the surface, creating a stabilizing feedback. If the dust flux rose, cryoconite was removed; if the dust flux waned, cryoconite accumulated. In addition to cyanobacteria, modern cryoconite holes are inhabited by green algae, fungi, protists, and certain metazoans. On Snowball Earth, cryoconite pans provided stable interconnected habitats for eukaryotes tolerant of fresh to brackish cold water on an ablation surface 60 million km² in area. Flushing and burial of organic matter was a potential source of atmospheric oxygen. Dominance of green algae among Ediacaran eukaryotic primary producers is a possible legacy of Cryogenian cryoconite pans, but a schizohaline ocean—supraglacial freshwater and subglacial brine—may have exerted selective stress on early metazoans, or impeded their evolution.     

Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.     

Changes in cell size during the cooling, warming and post-thawing periods of the freeze-thaw cycle.

Chinese Hamster Ovary (CHO) cells were cooled at 1 and 200 °C/min and subsequently thawed, while being studied with a cryomicroscope. Post-thaw size changes were measured with a Quantimet 720 Image Analysing Computer. It was found that the behavior of individuals in a population varied and depended on cooling rate. Cooling at 1 °C/min resulted in cells showing no intracellular ice, whereas cooling at 200 °C/min caused intracellular ice formation in some cells but not in others. In addition, at the slow rate, during cooling, the cells shrank significantly but swelled on thawing to become larger than non-frozen controls. Following swelling, as their temperature rose, the cells shrank to the size of non-frozen controls. At the fast rate, cells showed variation in their amount of intracellular ice and in their degree of shrinkage. Cells containing most ice shrank least. On warming, cells with intracellular ice began to swell at a lower temperature than did those cells without intracellular ice, while after thawing they swelled to a greater extent partly due to widespread blebbing. Corresponding recovery indices were measured, and correlation of these with the above effects suggests that: (i) cells completely filled with intracellular ice are non-viable; (ii) cells partially filled with intracellular ice respond to, or can be rescued by, first warming; (iii) cells without intracellular ice are viable; (iv) viable cells are those which regain their original size following thawing; (v) non-viable cells are those which remain swollen above their original size.     

Colonization and ecological development of new streams in Glacier Bay National Park, Alaska

SUMMARY 1. Colonization and ecological development of postglacial freshwater communities was investigated in Glacier Bay National Park, south-eastern Alaska, following the rapid recession of a Neo-glacial ice sheet within the last 250 years.
2. Environmental variables shown to be most significant in stream development were temperature, flow regime and sedimentation.
3. The Chironomidae (Diptera) were the pioneer invertebrate colonizers of newly emergent streams arising as meltwater from receding ice sheets and displayed a distinct pattern of succession with stream maturity.
4. Ephemeroptera and Plecoptera colonized warmer clearwater streams, but Trichoptera had a minimal role in invertebrate community development.
5. Establishment and production of salmonid fish populations in the new streams related principally to stream flow and sediment characteristics.
6. Future pathways along which the streams may develop is probably dependent on the degree of large organic debris input.
7. Stream development, structure and function are summarized including reference to theories of ecosystem development, ecological succession and community stability.     

III: Ultrastructure of Boar Spermatozoa Frozen Ultra-Rapidly at Various Stages of Conventional Freezing and Thawing

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

An isolated perfused rat mesentery model for direct observation of the vasculature during cryopreservation

An intact vasculature is essential for successful hypothermic perfusion and cryopreservation of solid organs, but few studies have specifically assessed the vascular effects of these procedures. A technique was therefore developed for continuous, direct observation of an isolated vascular bed during hypothermic perfusion with cryoprotectants, and during freezing and thawing. The isolated rat mesentery was spread across a controlled low temperature microscope stage and perfused with solutions containing fluorescein isothiocyanate (FITC)-Dextran 70 as an indicator of macromolecular permeability of the vessels. Hypertonic citrate washout, HP-5 perfusion (23), rapid and slow addition and removal of glycerol, and freezing/thawing were studied. Control perfusion with HP-5 produced slow FITC-Dextran leakage, reflecting normal physiological macromolecular permeability of vessels. Rapid addition of glycerol dramatically increased vascular permeability, consistent with osmotic damage to vessels. Rapid removal stopped flow through capillaries and decreased vascular dimensions, suggesting overhydration of endothelial cells and extravascular tissue. Venules and capillaries were the most susceptible vessels to osmotic stress. Slow addition and removal of glycerol (80 mmol/liter/min) produced results similar to control perfusions. During slow freezing (0.5 degree C/min to -5 degrees C) extravascular ice compressing vessels was more obvious than intravascular ice. Glycerol afforded some protection to the microvasculature during freeze/thaw cycles since flow was reestablished in venules and arterioles after thawing, although FITC-Dextran leakage indicated that damage had occurred.     

Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi

One hundred and ninety five strains of fungi were observed during freezing and thawing using a cryogenic light microscope. There was no obvious link between taxonomic position and their morphological response to freezing and thawing. The viability of seven of these strains was examined following freezing and thawing in the presence or absence of the cryoprotectants glycerol and dimethyl sulphoxide. Intracellular ice and hyphal shrinkage were not necessarily lethal events, but in many cases they affected the rate and quality of growth. Both cryoprotectants reduced shrinkage, shifted the cooling rate where intracellular ice formed in many cases, and improved the recovery of strains. The results presented aid the development of successful cryopreservation protocols.     

Late glacial and Holocene environmental changes inferred from sediments in Lake Myklevatnet,Nordfjord, western Norway

Late Glacial and Holocene environmental changes were reconstructed using physical, chemical and biological proxies in Lake Myklevatnet, Allmenningen, (5º13′17″E, 61º55′13″N) located at the northern side of Nordfjorden at the coast of western Norway. Myklevatnet (123 m a.s.l.) lies above the Late Glacial marine limit and contains sediments back to approximately 14,300 years before a.d. 2000 (b2k). Because the lake is located ~48 km beyond the margin of the Younger Dryas (YD) fjord and valley glaciers further inland, and did not receive glacier meltwater from local glaciers during the YD, the lake record provides supplementary information to Lake Kråkenes that received glacial meltwater from a local YD glacier. Lake Myklevatnet has a small catchment and is sensitive to Late Glacial and Holocene climate and environmental changes in the coastal region of western Norway. The age-depth relationship was inferred from a radiocarbon- and tephra-based smoothing-spline model with correlated ages from oxygen isotope maxima and minima in the Late Glacial sequence of the NGRIP ice core (in years b2k) to refine the basal chronology in the Myklevatnet record. The results indicate a two-step YD warming, colder early YD temperatures than in the later part of the YD, and considerably more climate and environmental variability during the late Holocene in western Norway than recorded previously in the oxygen isotopes from Greenland ice cores. The Myklevatnet record is also compared with other Late Glacial and Holocene terrestrial and marine proxy reconstructions in the North Atlantic realm.     

Glacial microbiota are hydrologically connected and temporally variable

Glaciers are melting rapidly. The concurrent export of microbial assemblages alongside glacial meltwater is expected to impact the ecology of adjoining ecosystems. Currently, the source of exported assemblages is poorly understood, yet this information may be critical for understanding how current and future glacial melt seasons may influence downstream environments. We report on the connectivity and temporal variability of microbiota sampled from supraglacial, subglacial and periglacial habitats and water bodies within a glacial catchment. Sampled assemblages showed evidence of being biologically connected through hydrological flowpaths, leading to a meltwater system that accumulates prokaryotic biota as it travels downstream. Temporal changes in the connected assemblages were similarly observed. Snow assemblages changed markedly throughout the sample period, likely reflecting changes in the surrounding environment. Changes in supraglacial meltwater assemblages reflected the transition of the glacial surface from snow-covered to bare-ice. Marked snowmelt across the surrounding periglacial environment resulted in the flushing of soil assemblages into the riverine system. In contrast, surface ice within the ablation zone and subglacial meltwaters remained relatively stable throughout the sample period. Our results are indicative that changes in snow and ice melt across glacial environments will influence the abundance and diversity of microbial assemblages transported downstream.     

Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

Ultrastructural observations on the frozen state of pancreatic acinar cells were correlated with results of parallel studies before freezing and after thawing, as to cryoinjury and cryoprotection.Data support an hypothesis of freezing injury based upon intracellular ice and solution effects during rapid and slow freezing, respectively. The basis for superiority of extracellular over intracellular glycerol in cryoprotection was demonstrated in terms of these factors.Evidence is offered to explain the ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum (RER) seen after thawing, relative to the combined effects of freezing rate and glycerol. Slow freezing, in combination with the presence of extracellular glycerol, provided sufficient dehydration to almost completely suppress intracellular ice formation, yielding minimal ultrastructural alteration of RER. Greatest cryoinjury, expressed as extensive conversion of RER into sphere-like vesicles, was induced by the extensive intracellular ice formation which accompanied rapid freezing. A mechanism is suggested to explain physical damage of RER by intracellular ice.     

Vulnerability of alpine stream biodiversity to shrinking glaciers and snowpacks

Climate change poses a considerable threat to the biodiversity of high latitude and altitude ecosystems, with alpine regions across the world already showing responses to warming. However, despite probable hydrological change as alpine glaciers and snowpacks shrink, links between alpine stream biota and reduced meltwater input are virtually unknown. Using data from the French Pyrénées, we demonstrate that taxonomic richness and total abundance of stream macroinvertebrates increase significantly as meltwater (snow melt and glacier melt) contributions to river flow decrease. Macroinvertebrate species showed a gradation of optimum meltwater conditions at which they persist. For example: Habroleptoides berthelemyi (Ephemeroptera), Perla grandis (Plecoptera) and Rhithrogena spp. (Ephemeroptera) increased in abundance when meltwater contributions to streamflow decrease, whereas in contrast, Rhyacophila angelieri (Trichoptera) and Diamesa latitarsis spp. (Diptera) decreased in abundance. Changes in alpine stream macroinvertebrate community composition as meltwater contributions decline were associated with lower suspended sediment concentration, and higher water temperature, electrical conductivity and pH. Our results suggest α diversity (at a site) of streams presently fed by meltwaters will increase with future meltwater reductions. However, β diversity (between‐sites) will be reduced as snow melt and glacier melt decrease because the habitat heterogeneity associated with spatiotemporal variability of water source contributions will become lower as meltwater contributions decline. Extinction of some endemic alpine aquatic species (such as the Pyrenean caddis fly R. angelieri) is predicted with reduced meltwater inputs, leading to decreases in γ diversity (region). Our identification of significant links between meltwater production and stream macroinvertebrate biodiversity has wider implications for the conservation of alpine river ecosystems under scenarios of climate change induced glacier and snowpack loss.     

Seasonal shifts in ice edge phytoplankton blooms in the Barents Sea related to the water column stability

The development of the phytoplankton bloom and its relation to water column stabilisation during the transition from early to high summer (of 1991) in the seasonally ice-covered zone of the Barents Sea were studied from a meridional transect of repeated hydrographic/biological stations. The water column stabilisation is described in detail with the aid of vertical profiles of the Brunt-Väisälä frequency squared (N²). The contributions of seasonal warming and ice melting to stabilisation are elucidated by determining the effects of temperature and salinity on N². The spring bloom in 1991 migrated poleward from June to July by about 400 km, associated with the retreat of the ice edge. The spring bloom culminated with maximum chlorophyll concentrations in the mixed layer about 100–300 km north of the centre of the meltwater lens, at its northern edge, where the ice cover was still substantial. From the distribution of N² it becomes obvious that the bloom starts at the very beginning of stabilisation, which results solely from the release of meltwater. The increase in temperature due to the seasonal warming does not contribute to the onset of vernal blooming; temperature starts to contribute to the stratification later, when the spring bloom has ceased due to the exhaustion of nutrients in the mixed layer. By that time a deep chlorophyll maximum has formed in the seasonal pycnocline, 20–30 m below the base of the mixed layer. The effect of the seasonal ice cover on the mean areal new primary production is discussed.     

Intracellular ice formation and growth in MCF-7 cancer cells

Direct cell injury in cryosurgery is highly related to intracellular ice formation (IIF) during tissue freezing and thawing. Mechanistic understanding of IIF in tumor cells is critical to the development of tumor cryo-ablation protocol. In aid of a high speed CMOS camera system, the events of IIF in MCF-7 cells have been studied using cryomicroscopy. Images of ‘darkening’ type IIF and recrystallization are compared between cells frozen with and without ice seeding. It is found that ice seeding has significant impact on the occurrence and growth of intracellular ice. Without ice seeding, IIF is observed to occur over a very small range of temperature (∼1 °C). The crystal dendrites are indistinguishable, which is independent of the cooling rate. Ice crystal grows much faster and covers the whole intracellular space in comparison to that with ice seeding, which ice stops growing near the cellular nucleus. Recrystallization is observed at the temperature from −13 °C to −9 °C during thawing. On the contrary, IIF occurs from −7 °C to −20 °C with ice seeding at a high subzero temperature (i.e., −2.5 °C). The morphology of intracellular ice frozen is greatly affected by the cooling rate, and no ‘darkening’ type ice formed inside cells during thawing. In addition, the intracellular ice formation is directional, which starts from the plasma membrane and grows toward the cellular nucleus with or without ice seeding. These results can be used to explain some findings of tumor cryosurgery in vivo, especially the causes of insufficient killing of tumor cells in the peripheral area near vessels.