Measuring Ice Crystal Growth: Methods and Insights

1. Frozen? How to Check! A Tutorial On Some Of The Current Methods Employed To Measure Ice Crystal Growth

2. Hexagonal Ice unit cell

3. Fun Facts • 275 (±25) water molecules are necessary to nucleate ice. • Ice nucleation is greatly enhanced (1010) at the air/water interface than in bulk ice. • Crystal growth is limited by the rate of diffusion at a low degree of supercooling (i.e < 2 °C; giving rise to more branching) but limited by the kinetics of growth at higher degrees of supercooling (i.e. > 4 °C; giving rise to needle-like growth). • Ice crystals are very pure and cannot incorporate solutes larger than helium. Some Chaotropic ions can be incorporated; Cl-, NH4+

4. Before we begin… Several general methods, experiments and assays exist, but each researcher will have their own variation and procedure for analysing results. This makes it very difficult to compare results and draw conclusions on efficacy.

5. Measuring and Studying Nucleation Homogenous nucleation occurs between -40 to -130°C, so most assays look at heterogeneous nucleation, or the effect of additives. Inhibition of bacterial ice nucleation by polyglycerol polymersBrian Wowk* and Gregory M. Fahy Cryobiology 44 (2002) 14–23 Several 1µL droplets in a DSC Sample pan, covered with oil Repeat for 100 samples

6. Nucleation 2. Large Volume Testing Fig. 4. Vials containing 15 g of 55% ethylene glycol and 1 ppm freeze-dried P. Syringaein water cooled to)-130 C. From left to right, the vials contain the additives 1% polyethylene glycol 1000 (control), 1% PVA, 1% PGL, and 0.5% PVA + 0.5% PGL.. Five-hundred-gram ethylene glycol (EG) solutions cooled to -128 C. The difference between (a) and (b) demonstrates the effect of water purity on ice crystal nucleation. The remaining solutions were prepared using ultrapure water with ice blocking polymers added as indicated. In solutions containing both polymers, the number of small vs large ice balls can be shifted by changing the relative balance of each polymer.

7. Nucleation 3. Gibson Group Method Microscope slide with 0.5 µL Droplets, maximum 6. Into cryostage, rapidly freeze, thaw, cool at 2K/min until all droplets have frozen, noting freezing temp for each droplet

8. Nucleation 4. DSC Microscopy Mashup Hai-Yan Wang, Takaaki Inada, Kunio Funakoshi, Shu-Shen Lu Cryobiology 59 (2009) 83–89 Melting temperatures DSC freezing temp % Ice fraction Residual solution concentration

9. Recrystallisation Practically, stopping ice growth is more useful and far easier than supressing nucleation events, as water containing solutions can be rapidly cooled, but thawing leads to large ice crystals and mechanical damage.

10. Recrystallisation 1. The ‘Splat’ Assay

11. Recrystallisation 2. Growth in Vitreous solution Hai-Yan Wang, Takaaki Inada, Kunio Funakoshi, Shu-Shen Lu Cryobiology 59 (2009) 83–89

12. Recrystallisation 3. Capillary Method This is a great method for long term measurements. The researchers typically tested samples after 16 hours. “The capillary method has several advantages over the splat or modified splat assays. First, a series of samples can be viewed and evaluated side-by-side, which aids in determining the endpoint of RI activity. With the splat method, each individual coverslip must be photographed separately and, only after the photographs or images are assembled and analyzed, can an endpoint be determined.” Melanie M Tomczak, Christopher B Marshall, Jack A Gilbert, Peter L Davies Biochemical and Biophysical Research Communications Volume 311, Issue 4, 28 November 2003, Pages 1041–1046 Parallel testing of a range of concentrations

13. Recrystallisation 4. DSC By examining the shape of DESC curve you can extract lots of useful data about the rate of crystal growth, and get a more complete picture of nucleation and growth. Crystallization is assumed to begin at point I, which is preceded by a short period, presumably due to the required thermal equilibration. An increasing heat flow due to the evolution of the enthalpy of crystallization is evident until a maximum is observed at point II. The rate of evolution of the enthalpy of crystallization depends strongly on the kinetics of the crystallization process. After point II, crystallization slows down significantly, and the measurement is terminated (i.e., at point III), where no noticeable change in the heat flow is further detected. Eti Baruch, Yitzhak Mastai, Macromolecular Rapid Communications Volume 28, Issue 23

14. Ice Shaping 1. Microscopy of single crystals Very fiddly and time consuming, but great results and images are possible. Its highly technical nature does not appeal to many researchers, so lots of alternatives. 1a. Ice is nucleated then slowly warmed until a single crystal is left, then the temperature slightly reduced and the morphology of the crystal after a stated time period is recorded. 1b. A tiny droplet of oil is suspended in a specialised cryostage. Into this droplet an even tinier droplet of water is added. The stage is cooled and the droplet freezes into (hopefully) a single crystal.

15. Ice Shaping 2. Templating Methods More obscure and only tends to be used with more exotic antifreeze agents, as the conditions are generally too harsh for antifreeze proteins. Generally, a suspension of alumina/PTFE/Silicon Carbide in A solution of the antifreeze agent is frozen, then the sample Is freeze dried and then kilned at 1350°C. The resulting structures can then be examined by SEM.

16. Ice Shaping 3. XRD (A) pure ice and (B) PEG113-b- PEI23-Gly solution.

17. Thermal Hysteresis via Gold Nanoparticles Frozen assembly of gold nanoparticles for rapid analysis of antifreeze protein activity, Young Pil Kim Biosensors and Bioelectronics, Volume 41, 15 March 2013, Pages 752–757 Seem to work well for measuring antifreeze protein thermal hysteresis activity. The other, and by far the most common method is to use DSC. Older methods exist, based on simple observation, but its easier to get a machine to do it now.

18. Resources • http://www1.lsbu.ac.uk/water/ • Ice Recrystallization Inhibitors: From Biological Antifreezes to Small Molecules, Chantelle J. Capicciotti, Malay Doshi and Robert N. Ben

19. Thanks Everyone!

20. Did I Miss Anything?