4 Notes
1. We attempted to perform a 10 μL drop of 40% sucrose to
obtain the same cooling rate; however, this was not feasible as
an opaque wafer formed (due to the highly concentrated solution) upon annealing at À6.4
C. We also attempted to sandwich a 10 μL drop between two coverslips and cool the sample
at a rate of 60
C/min on the aluminum block, but the liquid
could not be contained between the two coverslips as a result of
its relatively high volume.
2. It is worth noting that irregular-shaped ice crystals were
observed when a PBS solution was used in the sucrose assay
(SSA) and the ice crystals’ shapes were very inconsistent
between trials (see Fig. 1c).
3. For example, in the case of cryopreservation applications, frozen samples are generated resulting in a lower unfrozen fraction; therefore, the splat cooling assay would provide a more
accurate analysis of the efficiency of the IRI in that system. In
contrast, for applications in frozen foods, such as additives to
ice cream, the sucrose sandwich method would provide a more
accurate quantitative analysis for that application due to the
high sugar content in ice cream. Using IRI concentrations
based off of the splat cooling method would result in underestimated concentrations, and the IRI would not have its optimal effect in the system.
4. Coolant in cooling bath is prepared using 99% ethanol and
ethylene glycol (50:50 mixture).
5. This can be done by standing on a step ladder.
6. To prevent the formation of condensation on the coverslip,
wipe the area around the wafer using a Kimwipe.
References
1. Budke C, Heggemann C, Koch M, Sewald N,
Koop T (2009) Ice recrystallization kinetics in
the presence of synthetic antifreeze glycoprotein analogues using the framework of LSW
Theory. J Phys Chem B 113:2865–2873
2. Alley R, Perepezko J, Bentley C (1986) Grain
growth in polar ice: I. Theory. J Glaciol
32:415–424
3. Alley R, Perepezko J, Bentley C (1986) Grain
growth in polar ice: II. Application. J Glaciol
32:425–433
4. Sutton R, Lips A, Piccirillo G, Sztehlo A
(1996) Kinetics of ice recrystallization in aqueous fructose solutions. J Food Sci 61:741–745
5. Meryman H (1957) Physical limitations of the
rapid freezing method. Proc R Soc Lond A
147:452–459
6. Lovelock J, Bishop M (1959) Prevention of
freezing damage to living cells by dimethyl
sulphoxide. Nature 183:1394–1395
7. Lovelock JE, Polge C (1954) The immobilization of spermatozoa by freezing and thawing
and the protective action of glycerol. Biochem
J 58:618–622
8. McGann L (1978) Differing actions of
penetrating and nonpenetrating cryoprotective
agents. Cryobiology 15:382–390
9. Briard J, Jahan S, Chandran P, Allan D,
Pineault N, Ben R (2016) Small-molecule ice
280
Anna A. Ampaw et al.
1. We attempted to perform a 10 μL drop of 40% sucrose to
obtain the same cooling rate; however, this was not feasible as
an opaque wafer formed (due to the highly concentrated solution) upon annealing at À6.4
C. We also attempted to sandwich a 10 μL drop between two coverslips and cool the sample
at a rate of 60
C/min on the aluminum block, but the liquid
could not be contained between the two coverslips as a result of
its relatively high volume.
2. It is worth noting that irregular-shaped ice crystals were
observed when a PBS solution was used in the sucrose assay
(SSA) and the ice crystals’ shapes were very inconsistent
between trials (see Fig. 1c).
3. For example, in the case of cryopreservation applications, frozen samples are generated resulting in a lower unfrozen fraction; therefore, the splat cooling assay would provide a more
accurate analysis of the efficiency of the IRI in that system. In
contrast, for applications in frozen foods, such as additives to
ice cream, the sucrose sandwich method would provide a more
accurate quantitative analysis for that application due to the
high sugar content in ice cream. Using IRI concentrations
based off of the splat cooling method would result in underestimated concentrations, and the IRI would not have its optimal effect in the system.
4. Coolant in cooling bath is prepared using 99% ethanol and
ethylene glycol (50:50 mixture).
5. This can be done by standing on a step ladder.
6. To prevent the formation of condensation on the coverslip,
wipe the area around the wafer using a Kimwipe.
References
1. Budke C, Heggemann C, Koch M, Sewald N,
Koop T (2009) Ice recrystallization kinetics in
the presence of synthetic antifreeze glycoprotein analogues using the framework of LSW
Theory. J Phys Chem B 113:2865–2873
2. Alley R, Perepezko J, Bentley C (1986) Grain
growth in polar ice: I. Theory. J Glaciol
32:415–424
3. Alley R, Perepezko J, Bentley C (1986) Grain
growth in polar ice: II. Application. J Glaciol
32:425–433
4. Sutton R, Lips A, Piccirillo G, Sztehlo A
(1996) Kinetics of ice recrystallization in aqueous fructose solutions. J Food Sci 61:741–745
5. Meryman H (1957) Physical limitations of the
rapid freezing method. Proc R Soc Lond A
147:452–459
6. Lovelock J, Bishop M (1959) Prevention of
freezing damage to living cells by dimethyl
sulphoxide. Nature 183:1394–1395
7. Lovelock JE, Polge C (1954) The immobilization of spermatozoa by freezing and thawing
and the protective action of glycerol. Biochem
J 58:618–622
8. McGann L (1978) Differing actions of
penetrating and nonpenetrating cryoprotective
agents. Cryobiology 15:382–390
9. Briard J, Jahan S, Chandran P, Allan D,
Pineault N, Ben R (2016) Small-molecule ice
280
Anna A. Ampaw et al.