the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Micro-PINGUIN: Microtiter plate-based ice nucleation detection in gallium with an infrared camera
Abstract. Ice nucleation particles play a crucial role in atmospheric processes e.g., they can trigger ice formation in clouds and thus influence their lifetime and optical properties. The quantification and characterization of these particles require reliable and precise measurement techniques. In this publication, we present a novel droplet freezing instrument to measure immersion freezing of biotic and abiotic ice-nucleating particles within the temperature range of 0 °C to -25 °C. Immersion freezing of the samples is investigated using 384-well PCR plates with a sample volume of 30 µl. Nucleation events are detected with high precision using a thermal camera that records the increase in infrared emission due to the latent heat release. To maximize the thermal contact between the PCR plate and the surrounding cooling unit, we use a gallium bath as a mount for the PCR plate. The combination of good thermal connectivity and precise temperature recording enables accurate (± 0.81 °C at -10 °C) and reproducible (± 0.20 °C) detection of the nucleation temperatures.
For comparison with already existing instruments, the new ice nucleation instrument, “micro-PINGUIN”, was characterized using Snomax® (hereafter Snomax) and Illite NX suspensions. The results are in agreement with what has been reported in the literature for the already existing instruments. Consequently, the results that will be produced using the micro-PINGUIN are of good quality and can be compared to the results produced by other validated instruments for the study of immersion freezing of various ice nucleating particles. Further, we investigated the reproducibility of experiments using Snomax suspensions and found poor reproducibility when suspensions were prepared freshly even if the same batch of Snomax is used. This could be attributed to substrate heterogeneity, aging effects, and dilution errors. The reproducibility of the measurements is greatly improved for Snomax suspensions that are prepared in advance and stored frozen in aliquots. Thus, we suggest the use of suspensions frozen in aliquots for further reproducibility measurements and intercomparison studies.
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Notice on discussion status
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
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Preprint
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The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.
Journal article(s) based on this preprint
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2024-171', Anonymous Referee #1, 02 Feb 2024
In the manuscript submitted by Wieber et al., the authors developed a drop-freezing-based device designed for the investigation of ice nucleating particles via immersion freezing. While the instrument introduces innovations such as an infrared camera and a gallium substrate, its novelty is constrained when considering the extensive utilization of drop-freezing measurements. The temperature uncertainties of this device are well-presented and it is evaluated by measuring the ice nucleation activities of Snomax and illite NX, with comparison to results from the literature. The overall conclusion appears sound based on the presented results. Some statements are not well supported and some experimental details are missing. Specific comments are listed as follows.
Major comments:
- The authors recommend storing Snomax suspensions in aliquots. While I agree that the ice nucleation activity (INA) of Snomax suspensions with such a treatment shows improved reproducibility among repeating experiments. It’s noted that its INA has a lower value compared to the freshly prepared samples (Figure 5). This indicates that the sample experiences changes during storage. Therefore, the statement that aliquot storage is a better treatment for preserving Snomax samples requires further justification.
- The authors claim that the INAs of illite NX obtained in the present studies are comparable to those reported by previous studies. However, a difference in INAs obtained between the two datasets can reach up to 3 orders of magnitude (Figure 6). This conclusion on “The micro-PINGUIN instrument was validated using the well-studied substances Snomax and Illite NX and the results obtained in this study are consistent with already existing instruments.” needs justification.
- The temperature bias introduced by the inherent uncertainties from Pt 100 is not indicated in the present work.
- Specific details are absent in the schematic of the experimental setup, as outlined below.
Minor comments:
- Comments on figures: (1) For all figures: The name of each component can be indicated in the figure for clarity reasons. (2) Figure 1: Components E and D are not connected to any other units, can you make it more specific? If B is “water cooling”, how come the temperature of B goes to -2 oC? (3) Figure 2: What are the red and blue bars, current or circulating cooling fluid? (4) Figure 3: Can you also specify the flow rate in the legend? (5) Figure 7: clarify the meaning of horizontal error bars – whether they represent standard deviations or the previously mentioned temperature uncertainties.
- L170: What do you mean by “dry air”? What are the components of the dry air and how was it produced? Why the largely deviated freezing curve was not observed in other studies, most of which are used under room temperature and relative humidity conditions? Do you have any films to isolate the suspensions and air?
- L259: Do you mean the thermal conductivity between gallium and PCR trays?
- L125: Ensure consistency in referring to the "temperature probe" by specifying if it corresponds to the "pt100 probe" throughout the main text.
- Figure 3: Will higher flow rates>20 L/min introduce any changes in the freezing curve? I wonder if 20 L/min represents the optimal condition for your measurement.
- L352: Define "type A and type C INPs" for better comprehension. Provide an explanation for these terms.
Citation: https://doi.org/10.5194/egusphere-2024-171-RC1 -
AC1: 'Reply on RC1', Corina Wieber, 08 Mar 2024
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-171/egusphere-2024-171-AC1-supplement.pdf
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RC2: 'Comment on egusphere-2024-171', Anonymous Referee #2, 08 Feb 2024
The manuscript presents an instrument for quantifying ice nucleating particles active between 0 °C and -25 °C. Several similar instruments have been described before. Still, the introduction of an easily mouldable gallium block to hold various kinds of PCR plates is an innovation. Yet, infrared cameras have been used before to detect nucleation events (e.g., Kunert et al., 2018, https://doi.org/10.5194/amt-11-6327-2018). While accuracy and precision of the temperature measurement are thorough, the validation with two standard substances (snomax and illite) is somewhat limited by the variable freezing properties of suspensions prepared with these two substances. Perhaps birch pollen washing water, freezing within a particularly narrow temperature window from -17 °C to -18 °C (Häusler et al., 2018, https://www.mdpi.com/2073-4433/9/4/140), would have been a good third substance to test.
A total of 384 droplets provides the opportunity to derive differential freezing spectra (Vali, 2019, https://doi.org/10.5194/amt-12-1219-2019) that eventually show the different types of INP discussed in Section 3.3. With little effort a re-analysis of the available data may thereby yield additional insights into ice nucleation active components of snomax and illite that can hardly be gleaned from the cumulative spectra, e.g., in Figures 5, 6, 7, S4, and S5.
The infrared camera is said to detect the moment of an 'ice nucleation event' (line 68), whereas an optical camera observes a prolonged period, that is the 'change in optical properties such as brightness of the sample during the process of the whole droplet freezing' (lines 68 and 69). Right, but the 'ice nucleation event' can nevertheless be located in time at the beginning of changes in optical properties, no matter how long it took until the droplet was completely frozen. Perhaps I am wrong here, but I would expect to see in the infrared camera record at a warm freezing temperature, say at -4 °C, a rise in droplet temperature that is not a sudden step change and that also leaves some room for interpretation regarding the exact onset of freezing. The temperature record shown in Figure 4 is hard to analyse in this respect. Could you please show instead the record of a droplet frozen near -4 °C, and narrow the range of the time axis to the minute or so in which the peak occurred?
Lines 36 and 55: Replace 'high fraction' with 'large fraction' and 'high number' with 'large number'.
Line 56: Replace 'low number' with 'small number'.
Lines 90 to 92: Consider rearranging the sentence in this way: 'Furthermore, we address the challenges due to inhomogeneities of the product and due to aging effects and propose a possible solution for using Snomax as a suspension for intercomparison studies and reproducibility measurements.'
Figure 2: Better use a colour for the vapour chamber (G) that is different from that of the copper components above and below it.
Line 150 onwards: I appreciate the idea to heat the samples for repeated analysis, but how can evaporative loss be prevented, especially in heat treatments near boiling point?
The same question about evaporation arises in the next section, where the flow of dry air is discussed. During the development of the procedure, were sample trays weighed before and after a 40 min run to assess the loss due to evaporation?
Line 175: A 'was' is missing before 'usually'.
I am not sure whether Section 2.4 is needed because it mostly describes common practice. Consider reducing it to the bare minimum and merging it with the preceding section.
Figure 5a: At T > -11 °C error bars extend to 1 INP/mg snomax, suggesting that one of the three experiments no or very little freezing events were observed > -11 °C. After looking at Figure S4, I understand this is an artefact caused by the assumption of a normal distribution. In principle, you could estimate the multiplicative standard deviation (Limpert et al., 2001, https://academic.oup.com/bioscience/article/51/5/341/243981). However, three replicates cannot provide an estimate for that. Therefore, better show in Figure 5 all three replicates and not their mean and normal standard deviation.
Line 321: Consider to replace 'using' with 'analysing'.
Line 330: The statement 'are within the range of the concentrations reported therein' has to be narrowed to the temperature range in which it actually applies (-8 °C to -23 °C).
Figure S5: For fresh and old samples use better distinguishable symbols, e.g. , open circles and crosses, respectively.
Lines 376 and 377: Maybe reconsider the statement 'recognition of nucleation events instead of freezing events' (please see my earlier comment above on this issue).
Line 380: Not sure what is meant by 'intercomparable' here, perhaps 'comparable'?
Citation: https://doi.org/10.5194/egusphere-2024-171-RC2 -
AC2: 'Reply on RC2', Corina Wieber, 08 Mar 2024
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-171/egusphere-2024-171-AC2-supplement.pdf
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AC2: 'Reply on RC2', Corina Wieber, 08 Mar 2024
Interactive discussion
Status: closed
-
RC1: 'Comment on egusphere-2024-171', Anonymous Referee #1, 02 Feb 2024
In the manuscript submitted by Wieber et al., the authors developed a drop-freezing-based device designed for the investigation of ice nucleating particles via immersion freezing. While the instrument introduces innovations such as an infrared camera and a gallium substrate, its novelty is constrained when considering the extensive utilization of drop-freezing measurements. The temperature uncertainties of this device are well-presented and it is evaluated by measuring the ice nucleation activities of Snomax and illite NX, with comparison to results from the literature. The overall conclusion appears sound based on the presented results. Some statements are not well supported and some experimental details are missing. Specific comments are listed as follows.
Major comments:
- The authors recommend storing Snomax suspensions in aliquots. While I agree that the ice nucleation activity (INA) of Snomax suspensions with such a treatment shows improved reproducibility among repeating experiments. It’s noted that its INA has a lower value compared to the freshly prepared samples (Figure 5). This indicates that the sample experiences changes during storage. Therefore, the statement that aliquot storage is a better treatment for preserving Snomax samples requires further justification.
- The authors claim that the INAs of illite NX obtained in the present studies are comparable to those reported by previous studies. However, a difference in INAs obtained between the two datasets can reach up to 3 orders of magnitude (Figure 6). This conclusion on “The micro-PINGUIN instrument was validated using the well-studied substances Snomax and Illite NX and the results obtained in this study are consistent with already existing instruments.” needs justification.
- The temperature bias introduced by the inherent uncertainties from Pt 100 is not indicated in the present work.
- Specific details are absent in the schematic of the experimental setup, as outlined below.
Minor comments:
- Comments on figures: (1) For all figures: The name of each component can be indicated in the figure for clarity reasons. (2) Figure 1: Components E and D are not connected to any other units, can you make it more specific? If B is “water cooling”, how come the temperature of B goes to -2 oC? (3) Figure 2: What are the red and blue bars, current or circulating cooling fluid? (4) Figure 3: Can you also specify the flow rate in the legend? (5) Figure 7: clarify the meaning of horizontal error bars – whether they represent standard deviations or the previously mentioned temperature uncertainties.
- L170: What do you mean by “dry air”? What are the components of the dry air and how was it produced? Why the largely deviated freezing curve was not observed in other studies, most of which are used under room temperature and relative humidity conditions? Do you have any films to isolate the suspensions and air?
- L259: Do you mean the thermal conductivity between gallium and PCR trays?
- L125: Ensure consistency in referring to the "temperature probe" by specifying if it corresponds to the "pt100 probe" throughout the main text.
- Figure 3: Will higher flow rates>20 L/min introduce any changes in the freezing curve? I wonder if 20 L/min represents the optimal condition for your measurement.
- L352: Define "type A and type C INPs" for better comprehension. Provide an explanation for these terms.
Citation: https://doi.org/10.5194/egusphere-2024-171-RC1 -
AC1: 'Reply on RC1', Corina Wieber, 08 Mar 2024
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-171/egusphere-2024-171-AC1-supplement.pdf
-
RC2: 'Comment on egusphere-2024-171', Anonymous Referee #2, 08 Feb 2024
The manuscript presents an instrument for quantifying ice nucleating particles active between 0 °C and -25 °C. Several similar instruments have been described before. Still, the introduction of an easily mouldable gallium block to hold various kinds of PCR plates is an innovation. Yet, infrared cameras have been used before to detect nucleation events (e.g., Kunert et al., 2018, https://doi.org/10.5194/amt-11-6327-2018). While accuracy and precision of the temperature measurement are thorough, the validation with two standard substances (snomax and illite) is somewhat limited by the variable freezing properties of suspensions prepared with these two substances. Perhaps birch pollen washing water, freezing within a particularly narrow temperature window from -17 °C to -18 °C (Häusler et al., 2018, https://www.mdpi.com/2073-4433/9/4/140), would have been a good third substance to test.
A total of 384 droplets provides the opportunity to derive differential freezing spectra (Vali, 2019, https://doi.org/10.5194/amt-12-1219-2019) that eventually show the different types of INP discussed in Section 3.3. With little effort a re-analysis of the available data may thereby yield additional insights into ice nucleation active components of snomax and illite that can hardly be gleaned from the cumulative spectra, e.g., in Figures 5, 6, 7, S4, and S5.
The infrared camera is said to detect the moment of an 'ice nucleation event' (line 68), whereas an optical camera observes a prolonged period, that is the 'change in optical properties such as brightness of the sample during the process of the whole droplet freezing' (lines 68 and 69). Right, but the 'ice nucleation event' can nevertheless be located in time at the beginning of changes in optical properties, no matter how long it took until the droplet was completely frozen. Perhaps I am wrong here, but I would expect to see in the infrared camera record at a warm freezing temperature, say at -4 °C, a rise in droplet temperature that is not a sudden step change and that also leaves some room for interpretation regarding the exact onset of freezing. The temperature record shown in Figure 4 is hard to analyse in this respect. Could you please show instead the record of a droplet frozen near -4 °C, and narrow the range of the time axis to the minute or so in which the peak occurred?
Lines 36 and 55: Replace 'high fraction' with 'large fraction' and 'high number' with 'large number'.
Line 56: Replace 'low number' with 'small number'.
Lines 90 to 92: Consider rearranging the sentence in this way: 'Furthermore, we address the challenges due to inhomogeneities of the product and due to aging effects and propose a possible solution for using Snomax as a suspension for intercomparison studies and reproducibility measurements.'
Figure 2: Better use a colour for the vapour chamber (G) that is different from that of the copper components above and below it.
Line 150 onwards: I appreciate the idea to heat the samples for repeated analysis, but how can evaporative loss be prevented, especially in heat treatments near boiling point?
The same question about evaporation arises in the next section, where the flow of dry air is discussed. During the development of the procedure, were sample trays weighed before and after a 40 min run to assess the loss due to evaporation?
Line 175: A 'was' is missing before 'usually'.
I am not sure whether Section 2.4 is needed because it mostly describes common practice. Consider reducing it to the bare minimum and merging it with the preceding section.
Figure 5a: At T > -11 °C error bars extend to 1 INP/mg snomax, suggesting that one of the three experiments no or very little freezing events were observed > -11 °C. After looking at Figure S4, I understand this is an artefact caused by the assumption of a normal distribution. In principle, you could estimate the multiplicative standard deviation (Limpert et al., 2001, https://academic.oup.com/bioscience/article/51/5/341/243981). However, three replicates cannot provide an estimate for that. Therefore, better show in Figure 5 all three replicates and not their mean and normal standard deviation.
Line 321: Consider to replace 'using' with 'analysing'.
Line 330: The statement 'are within the range of the concentrations reported therein' has to be narrowed to the temperature range in which it actually applies (-8 °C to -23 °C).
Figure S5: For fresh and old samples use better distinguishable symbols, e.g. , open circles and crosses, respectively.
Lines 376 and 377: Maybe reconsider the statement 'recognition of nucleation events instead of freezing events' (please see my earlier comment above on this issue).
Line 380: Not sure what is meant by 'intercomparable' here, perhaps 'comparable'?
Citation: https://doi.org/10.5194/egusphere-2024-171-RC2 -
AC2: 'Reply on RC2', Corina Wieber, 08 Mar 2024
The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2024/egusphere-2024-171/egusphere-2024-171-AC2-supplement.pdf
-
AC2: 'Reply on RC2', Corina Wieber, 08 Mar 2024
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- 1
Corina Wieber
Mads Rosenhøj Jeppesen
Kai Finster
Claus Melvad
Tina Šantl-Temkiv
The requested preprint has a corresponding peer-reviewed final revised paper. You are encouraged to refer to the final revised version.