Abstract. The second phase of the Fifth International Ice Nucleation Workshop (FIN-02) involved the gathering of a large number of researchers at the Karlsruhe Institute of Technology's Aerosol Interactions and Dynamics of the Atmosphere (AIDA) facility to promote characterization and understanding of ice nucleation measurements made by a variety of methods used worldwide. Compared to the previous workshop in 2007, participation was doubled, reflecting a vibrant research area. Experimental methods involved sampling of aerosol particles by direct processing ice nucleation measuring systems from the same volume of air in separate experiments using different ice nucleating particle (INP) types, and collections of aerosol particle samples onto filters or into liquid for sharing amongst measurement techniques that post-process these samples. In this manner, any errors introduced by differences in generation methods when samples are shared across laboratories were mitigated. Furthermore, as much as possible, aerosol particle size distribution was controlled so that the size limitations of different methods were minimized. The results presented here use data from the workshop to assess the comparability of immersion freezing measurement methods activating INPs in bulk suspensions, methods that activate INPs in condensation and/or immersion freezing modes as single particles on a substrate, continuous flow diffusion chambers (CFDCs) directly sampling and processing particles well above water saturation to maximize immersion and subsequent freezing of aerosol particles, and expansion cloud chamber simulations in which liquid cloud droplets were first activated on aerosol particles prior to freezing. The AIDA expansion chamber measurements are expected to be the closest representation to INP activation in atmospheric cloud parcels in these comparisons, due to exposing particles freely to adiabatic cooling. The different particle types used as INPs included the minerals illite NX and potassium feldspar (K-feldspar), two natural soil dusts representative of arable sandy loam (Argentina) and highly erodible sandy dryland (Tunisia) soils, respectively, and a bacterial INP (Snomax®). Considered together, the agreement among post-processed immersion freezing measurements of the numbers and fractions of particles active at different temperatures following bulk collection of particles into liquid was excellent, with possible temperature uncertainties inferred to be a key factor in determining INP uncertainties. Collection onto filters for rinsing versus directly into liquid in impingers made little difference. For methods that activated collected single particles on a substrate at a controlled humidity at or above water saturation, agreement with immersion freezing methods was good in most cases, but was biased low in a few others for reasons that have not been resolved, but could relate to water vapor competition effects. Amongst CFDC-style instruments, various factors requiring (variable) higher supersaturations to achieve equivalent immersion freezing activation dominate the uncertainty between these measurements, and for comparison with bulk immersion freezing methods. When operated above water saturation to include assessment of immersion freezing, CFDC measurements often measured at or above the upper bound of immersion freezing device measurements, but often underestimated INP concentration in comparison to an immersion freezing method that first activates all particles into liquid droplets prior to cooling (the PIMCA-PINC device, or Portable Immersion Mode Cooling chAmber–Portable Ice Nucleation Chamber), and typically slightly underestimated INP number concentrations in comparison to cloud parcel expansions in the AIDA chamber; this can be largely mitigated when it is possible to raise the relative humidity to sufficiently high values in the CFDCs, although this is not always possible operationally. Correspondence of measurements of INPs among direct sampling and post-processing systems varied depending on the INP type. Agreement was best for Snomax® particles in the temperature regime colder than −10 ∘C, where their ice nucleation activity is nearly maximized and changes very little with temperature. At temperatures warmer than −10 ∘C, Snomax® INP measurements (all via freezing of suspensions) demonstrated discrepancies consistent with previous reports of the instability of its protein aggregates that appear to make it less suitable as a calibration INP at these temperatures. For Argentinian soil dust particles, there was excellent agreement across all measurement methods; measures ranged within 1 order of magnitude for INP number concentrations, active fractions and calculated active site densities over a 25 to 30 ∘C range and 5 to 8 orders of corresponding magnitude change in number concentrations. This was also the case for all temperatures warmer than −25 ∘C in Tunisian dust experiments. In contrast, discrepancies in measurements of INP concentrations or active site densities that exceeded 2 orders of magnitude across a broad range of temperature measurements found at temperatures warmer than −25 ∘C in a previous study were replicated for illite NX. Discrepancies also exceeded 2 orders of magnitude at temperatures of −20 to −25 ∘C for potassium feldspar (K-feldspar), but these coincided with the range of temperatures at which INP concentrations increase rapidly at approximately an order of magnitude per 2 ∘C cooling for K-feldspar. These few discrepancies did not outweigh the overall positive outcomes of the workshop activity, nor the future utility of this data set or future similar efforts for resolving remaining measurement issues. Measurements of the same materials were repeatable over the time of the workshop and demonstrated strong consistency with prior studies, as reflected by agreement of data broadly with parameterizations of different specific or general (e.g., soil dust) aerosol types. The divergent measurements of the INP activity of illite NX by direct versus post-processing methods were not repeated for other particle types, and the Snomax® data demonstrated that, at least for a biological INP type, there is no expected measurement bias between bulk collection and direct immediately processed freezing methods to as warm as −10 ∘C. Since particle size ranges were limited for this workshop, it can be expected that for atmospheric populations of INPs, measurement discrepancies will appear due to the different capabilities of methods for sampling the full aerosol size distribution, or due to limitations on achieving sufficient water supersaturations to fully capture immersion freezing in direct processing instruments. Overall, this workshop presents an improved picture of present capabilities for measuring INPs than in past workshops, and provides direction toward addressing remaining measurement issues.

Abstract. Birch pollen are known to release ice-nucleating macromolecules (INM), but little is known about the production and release of INM from other parts of the tree. We examined the ice nucleation activity of samples from 10 different birch trees (Betula spp.). Samples were taken from nine birch trees in Tyrol, Austria, and from one tree in a small urban park in Vienna, Austria. Filtered aqueous extracts of 30 samples of leaves, primary wood (new branch wood, green in colour, photosynthetically active), and secondary wood (older branch wood, brown in colour, with no photosynthetic activity) were analysed in terms of ice nucleation activity using VODCA (Vienna Optical Droplet Crystallization Analyser), a cryo microscope for emulsion samples. All samples contained ice-nucleating particles in the submicron size range. Concentrations of ice nuclei ranged from 6.7×104 to 6.1×109 mg−1 sample. Mean freezing temperatures varied between −15.6 and −31.3 ∘C; the range of temperatures where washes of birch pollen and dilutions thereof typically freeze. The freezing behaviour of three concentrations of birch pollen washing water (initial wash, 1 : 100, and 1 : 10 000) were significantly associated with more than a quarter of our samples, including some of the samples with highest and lowest activity. This indicates a relationship between the INM of wood, leaves, and pollen. Extracts derived from secondary wood showed the highest concentrations of INM and the highest freezing temperatures. Extracts from the leaves exhibited the highest variation in INM and freezing temperatures. Infrared spectra of the extracts and tested birch samples show qualitative similarity, suggesting the chemical components may be broadly similar.

Abstract. Within the last years pollen grains have gained increasing attention due to their cloud-forming potential. Especially the discovery that ice nucleating macromolecules (INMs) or subpollen particles (SPPs) obtained from pollen grains are able to initiate freezing has stirred up interest in pollen. INMs and SPPs are much smaller and potentially more numerous than pollen grains and could significantly affect cloud formation in the atmosphere. However, INMs and SPPs are not clearly distinguished. This has motivated the present study, which focuses on birch pollen and investigates the relationship between pollen grains, INMs, and SPPs. According to the usage of the term SPP in the medical fields, we define SPPs as the starch granules contained in pollen grains. We show that these insoluble SPPs are only obtained when fresh pollen grains are used to generate aqueous extracts from pollen. Due to the limited seasonal availability of fresh pollen grains, almost all studies have been conducted with commercial pollen grains. To enable the investigation of the SPPs we develop an alternative extraction method to generate large quantities of SPPs from commercial pollen grains. We show that INMs are not bonded to SPPs (i.e. can be washed off with water). Further, we find that purified SPPs are not ice nucleation active: after several times of washing SPPs with ultrapure water the ice nucleation activity completely disappears. To our knowledge, this is the first study to investigate the ice nucleation activity of isolated SPPs. To study the chemical nature of the INMs, we use fluorescence spectroscopy. Fluorescence excitation–emission maps indicate a strong signal in the protein range (maximum around λex = 280 nm and λem = 330 nm) with all ice nucleation active samples. In contrast, with purified SPPs the protein signal is lost. We also quantify the protein concentration with the Bradford assay. The protein concentration ranges from 77.4 µg mL−1 (highly concentrated INMs) to below 2.5 µg mL−1 (purified SPPs). Moreover, we investigate the connection between proteins and ice nucleation activity by treating the ice nucleation active samples with subtilisin A and urea to unfold and digest the proteins. After this treatment the ice nucleation activity clearly diminished. The results indicate a linkage between ice nucleation activity and protein concentration. The missing piece of the puzzle could be a glycoprotein which exhibits carboxylate functionalities, can bind water in tertiary structures, and displays degeneration and unfolding of its secondary structure due to heat treatment or reaction with enzymes. Even though purified SPPs are not ice nucleation active they could act as carriers of INMs and distribute those in the atmosphere.

New tools and technology are needed to study microorganisms in freshwater environments. Little is known about spatial distribution and ice nucleation activity (INA) of microorganisms in freshwater lakes. We developed a system to collect water samples from the surface of lakes using a 3D-printed sampling device tethered to a drone (DOWSE, DrOne Water Sampling SystEm). The DOWSE was used to collect surface water samples at different distances from the shore (1, 25, and 50 m) at eight different freshwater lakes in Austria in June 2018. Water samples were filtered, and microorganisms were cultured on two different media types, TSA (a general growth medium) and KBC (a medium semi-selective for bacteria in the genus Pseudomonas). Mean concentrations (colony forming units per mL, or CFU/mL) of bacteria cultured on TSA ranged from 19,800 (Wörthersee) to 210,500 (Gosaulacke) CFU/mL, and mean concentrations of bacteria cultured on KBC ranged from 2590 (Ossiachersee) to 11,000 (Vorderer Gosausee) CFU/mL. There was no significant difference in sampling distance from the shore for concentrations of microbes cultured on TSA (p = 0.28). A wireless bathymetry sensor was tethered to the drone to map temperature and depth across the sampling domain of each of the lakes. At the 50 m distance from the shore, temperature ranged from 17 (Hinterer Gosausee, and Gosaulacke) to 26 • C (Wörthersee), and depth ranged from 2.8 (Gosaulacke) to 11.1 m (Grundlsee). Contour maps of concentrations of culturable bacteria across the drone sampling domain revealed areas of high concentrations (hot spots) in some of the lakes. The percentage of ice-nucleation active (ice+) bacteria cultured on KBC ranged from 0% (0/64) (Wörthersee) to 58% (42/72) (Vorderer Gosausee), with a mean of 28% (153/544) for the entire sample set. Future work aims to elucidate the structure and function of entire microbial assemblages within and among the Austrian lakes.

Terrestrial ecosystems can influence atmospheric processes by contributing a huge variety of biological aerosols (bioaerosols) to the environment. Several types of biological particles, such as pollen grains, fungal spores, and bacteria cells, trigger freezing processes in super-cooled cloud droplets, and as such can contribute to the hydrological cycle. Even though biogenic particles are known as the most active form of ice nucleation particles (INPs), the transport to high tropospheric altitudes, as well as the occurrence in clouds, remains understudied. Thus, transport processes from the land surface into the atmosphere need to be investigated to estimate weather phenomena and climate trends. To help fill this knowledge gap, we developed a drone-based aerosol particles sampling impinger/impactor (DAPSI) system for field studies to investigate sources and near surface transport of biological INPs. DAPSI was designed to attach to commercial rotary-wing drones to collect biological particles within about 100 m of the Earth’s surface. DAPSI provides information on particulate matter concentrations (PM10 & PM2.5), temperature, relative humidity, and air pressure at about 0.5 Hz, by controlling electrical sensors with an onboard computer (Raspberry Pi 3). Two remote-operated sampling systems (impinging and impacting) were integrated into DAPSI. Laboratory tests of the impinging system showed a 96% sampling efficiency for standardized aerosol particles (2 µm polystyrene latex spheres) and 84% for an aerosol containing biological INPs (Betula pendula). A series of sampling missions (12 flights) were performed using two Phantom 4 quadcopters with DAPSI onboard at a remote sampling site near Gosau, Austria. Fluorescence microscopy of impactor foils showed a significant number of auto-fluorescent particles < 0.5 µm at an excitation of 465–495 nm and an emission of 515–555 nm. A slight increase in ice nucleation activity (onset temperature between −27 °C and −31 °C) of sampled aerosol was measured by applying freezing experiments with a microscopic cooling technique. There are a number of unique opportunities for DAPSI to be used to study the transport of bioaerosols, particularly for investigations of biological INP emissions from natural sources such as birch or pine forests.