No abstract

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Abstract. Ice-nucleating particles (INPs) are atmospheric aerosol particles that can strongly influence the radiative properties and precipitation onset in mixed-phase clouds by triggering ice formation in supercooled cloud water droplets. The ability to distinguish between INPs of mineral and biological origin in samples collected from the environment is needed to better understand their distribution and sources. A common method for assessing the relative contributions of mineral and biogenic INPs in samples collected from the environment (e.g. aerosol, rainwater, soil) is to determine the ice-nucleating ability (INA) before and after heating, where heat is expected to denature proteins associated with some biological ice nucleants. The key assumption is that the ice nucleation sites of biological origin are denatured by heat, while those associated with mineral surfaces remain unaffected; we test this assumption here. We exposed atmospherically relevant mineral samples to wet heat (INP suspensions warmed to above 90 ∘C) or dry heat (dry samples heated up to 250 ∘C) and assessed the effects on their immersion mode INA using a droplet freezing assay. K-feldspar, thought to be the dominant mineral-based atmospheric INP type where present, was not significantly affected by wet heating, while quartz, plagioclase feldspars and Arizona Test Dust (ATD) lost INA when heated in this mode. We argue that these reductions in INA in the aqueous phase result from direct alteration of the mineral particle surfaces by heat treatment rather than from biological or organic contamination. We hypothesise that degradation of active sites by dissolution of mineral surfaces is the mechanism in all cases due to the correlation between mineral INA deactivation magnitudes and their dissolution rates. Dry heating produced minor but repeatable deactivations in K-feldspar particles but was generally less likely to deactivate minerals compared to wet heating. We also heat-tested biogenic INP proxy materials and found that cellulose and pollen washings were relatively resistant to wet heat. In contrast, bacterially and fungally derived ice-nucleating samples were highly sensitive to wet heat as expected, although their activity remained non-negligible after wet heating. Dry heating at 250 ∘C leads to deactivation of all biogenic INPs. However, the use of dry heat at 250 ∘C for the detection of biological INPs is limited since K-feldspar's activity is also reduced under these conditions. Future work should focus on finding a set of dry heat conditions where all biological material is deactivated, but key mineral types are not. We conclude that, while wet INP heat tests at (>90 ∘C) have the potential to produce false positives, i.e. deactivation of a mineral INA that could be misconstrued as the presence of biogenic INPs, they are still a valid method for qualitatively detecting very heat-sensitive biogenic INPs in ambient samples if the mineral-based INA is controlled by K-feldspar.

Clouds containing supercooled water are important for both climate and weather, but our knowledge of which aerosol particle types nucleate ice in these clouds is far from complete. Combustion aerosols have strong anthropogenic sources, and if these aerosol types were to nucleate ice in clouds, they might exert a climate forcing. Here, we quantified the atmospheric ice-nucleating particle (INP) concentrations during the United Kingdom's annual Bonfire Night celebrations, which are characterized by large amounts of combustion aerosol from bonfires and fireworks. We used three immersion mode techniques covering more than 6 orders of magnitude in INP concentration over the temperature range from −10°C to homogeneous freezing. We found no observable systematic change in the INP concentration on three separate nights, despite more than a factor of 10 increase in aerosol number concentrations, up to a factor of 10 increase in PM 10 concentration, and more than a factor of 100 increase in black carbon (BC) mass concentration relative to pre-event levels. This implies that BC and other combustion aerosol such as ash did not compete with the INPs present in the background air. Furthermore, the upper limit of the ice-active site surface density, n s (T), of BC generated in these events was shown to be consistent with several other recent laboratory studies, showing a very low ice-nucleating activity of BC. We conclude that combustion aerosol particles similar to those emitted on Bonfire Night are at most of secondary importance for the INP population relevant for mixed-phase clouds in typical midlatitude terrestrial locations. Plain Language Summary Liquid water droplets found in clouds can cool to well below 0°C while remaining in the liquid phase (this is known as supercooling). These supercooled droplets can remain liquid down to below around −33°C without freezing, unless there is a certain type of aerosol particle present: an ice-nucleating particle (INP). Hence, INPs have the potential to drastically change the properties and lifetime of clouds, but the sources of INP in the atmosphere are poorly defined. In this study we measured the INP concentration before, during, and after a major combustion aerosol event in the United Kingdom, Bonfire Night. This celebration is characterized by bonfires (primarily made of waste wood, but also containing garden and household waste) and fireworks. We found that aerosol particles emitted during the celebration are not as effective at nucleating ice as aerosol particle already present in the atmosphere. We conclude that aerosol particles emitted from combustion processes such as those observed on Bonfire Night are not an important source of INPs.

Abstract. Ice-nucleating particles (INPs) are atmospheric aerosol particles that can strongly influence the radiative properties and precipitation onset in mixed-phase clouds by triggering ice formation in supercooled cloud water droplets. The ability to distinguish between INPs of mineral and biological origin in samples collected from the environment is needed to better understand their distribution and sources, but this is challenging. A common method for assessing the relative contributions of mineral and biogenic INPs in samples collected from the environment (e.g., aerosol, rainwater, soil) is to determine the ice-nucleating ability (INA) before and after heating, where heat is expected to denature proteins associated with biological ice nucleants. The key assumption is that the ice nucleation sites of biological origin are denatured by heat, while those associated with mineral surfaces remain unaffected; we test this assumption here. We exposed atmospherically relevant mineral samples to wet heat (INP suspensions warmed to above 90 °C) or dry heat (dry samples heated to 250 °C) and assessed the effects on their immersion mode INA using a droplet freezing assay. K-feldspar, thought to be the dominant mineral-based atmospheric INP type where present, was not significantly affected by wet heating, while quartz, plagioclase feldspars and Arizona test Dust (ATD) lost INA when heated in this mode. We argue that these reductions in INA in the aqueous phase result from direct alteration of the mineral particle surfaces by heat treatment rather than from biological or organic contamination. We hypothesise that degradation of active sites by dissolution of mineral surfaces is the mechanism in all cases due to the correlation between mineral INA deactivation magnitudes and their dissolution rates. Dry heating produced minor but repeatable deactivations in K-feldspar particles but was generally less likely to deactivate minerals compared to wet heating. We also heat tested proteinaceous and non-proteinaceous biogenic INP proxy materials and found that non-proteinaceous samples (cellulose and pollen) were relatively heat resistant. In contrast, the proteinaceous ice-nucleating samples were highly sensitive to wet and dry heat, as expected, although their activity remained non-negligible after heating. We conclude that, while INP heat tests have the potential to produce false positives, i.e., deactivation of a mineral INA that could be misconstrued as the presence of biogenic INPs, they are still a valid method for qualitatively detecting proteinaceous biogenic INP in ambient samples, so long as the mineral-based INA is controlled by K-feldspar.