Figure 1 shows a section of the spectra from m/z 288.5 to m/z 342.5 for the cell-free model biofilm sample 1 (top), a Bacillus subtilis biofilm from the study by Akbari et al. [4] (middle), and the ciprofloxacin-free control sample 3 (bottom). In the cell-free model biofilm, the ciprofloxacin and ice are uniformly distributed in the sample (see figure S3). All three spectra show the same pattern of peaks, which arise from water cluster ions. Peaks from ciprofloxacin can be seen in both the cell-free model biofilm and the B. subtilis biofilm, superimposed on the repeating water cluster pattern. All other peaks in this range of the spectrum arise from water cluster ions of the form (H2O)nX+ (henceforth cationized water clusters), where X+ is one of at least 18 different cations. In the ciprofloxacin-free control, peaks from CO2+ cationized water clusters are present at the [ciprofloxacin-OH]+ and [ciprofloxacin+H]+ masses. The spectra are shown on a log scale to allow better visualization of the smaller cationized water cluster peaks. The largest peaks from stable ions in all three spectra arise from protonated water cluster ions (H2O)nH+, which are labeled in Fig. 1. To the left of each of the protonated water cluster peaks are a pair of metastable peaks, which arise from ejection of a single H2O molecule from a higher mass protonated water cluster ion [9,10,11]. The red dots in Fig. 1 mark metastable peaks generated from the loss of H2O from an (H2O)18H+ parent ion. The metastable peaks were unambiguously identified by varying the analyzer voltages. There are two metastable peaks at each nominal mass arising from decays in the two field-free drift zones prior to the reflectron. A detailed description of the behavior of the metastable peaks has been described previously [9]. In the mass range shown, the metastable peaks are more intense than the stable water cluster ion peaks. Between the protonated water cluster ion peaks, there are peaks at every nominal mass. These peaks are from water cluster ions that contain a cation, such as (H2O)16NH4+, (H2O)16CH3+, and (H2O)15Na+ (plus at least 14 other cations) and peaks from the metastable decay of each of these ions. In ToF-SIMS, analysis of the metastable peaks can be used to unambiguously identify fragmentation pathways and verify molecular structure [11]. The metastable peaks associated with each of these cationized water clusters confirm that they are indeed from water cluster ions (9) (see figure S4 in the Appendix). There are at least 18 different cations in each water cluster sequence. The pattern shown in Fig. 1 between m/z 288.5 and m/z 305.5 is repeated every 18.01 u (the mass of an H2O) across the full mass spectrum from m/z 19 through m/z 2000, the highest mass measured.

ToF-SIMS spectra in the mass range of m/z 288.5 to m/z 342.5 from the cell-free model biofilm (top), a B. subtilis biofilm [4] treated with 1000 µg/ml ciprofloxacin (middle) and the ciprofloxacin-free control (bottom). The ciprofloxacin peaks are superimposed on a repeating pattern of cationized water clusters

The [ciprofloxacin+H]+ falls within a mass difference of 0.018 u to the (H2O)16CO2+ water cluster ion peak, which is unresolvable under the attainable mass resolution. At the high ciprofloxacin concentration used in this study, the (H2O)16CO2+ signal is more than two orders of magnitude lower than the [ciprofloxacin+H]+ signal, so the interference from the (H2O)16CO2+ water cluster is negligible. However, in the study by Akbari et al. [4], the (H2O)16CO2+ water cluster peak was found to prevent the detection of ciprofloxacin at concentrations below 10 µg/ml. In that study, the water cluster ions were also found to interfere with many other peaks commonly detected in bacterial biofilms.

A set of measurements was made on a ciprofloxacin-free control with cycled temperature variations. The data (see figure S5) reveal reversible effects below 153 K, so freeze-drying does not occur in the temperature range below 153 K. At a constant temperature of 163 K, the peak intensities of NH4+ cationized water clusters, smaller molecules, and elements, as well as the analyzer pressure changed significantly. Hence, we suspect that freeze-drying begins several degrees above 153 K, but below 163 K. At temperatures higher than 173 K, rapid freeze-drying is observed. This is consistent with temperature programmed ice measurements by Barros et al. [6, 7] where the ice thickness did not change until temperatures above 157 K.

The temperature trends for water cluster peaks that contain the same cation were found to be strongly correlated. Figure 2 shows the temperature trends for 6 of these cations for clusters in the mass range from m/z 180 to m/z 827 for the cell-free model biofilm (sample 1). For the protonated water cluster ions, this mass range corresponds to clusters that contain between 10 and 45 water molecules. The data shown in Fig. 2 was binned to 1 u so that metastable and stable peaks at the same nominal mass are combined. Furthermore, temperature trends were smoothed. Temperature trends for the full set of 18 cation masses are provided in the supplemental material figures S6, S7, and S8. The temperature trend for the total ion signal is shown in figure S9. One mass unit above the (H2O)n cluster masses, the protonated (X+ ⊃ H+ means X+ is a superset of H+) water cluster ion signals (Fig. 2, top left) are at least an order of magnitude higher than the other cationized water cluster ions. The signals from these ions remain approximately constant from 100 until 140 K and then decrease gradually until approximately 173 K when rapid freeze-drying of the sample begins. The intensity gap around 103 counts between the blue and green lines divides the protonated clusters (H2O)21H+ (m/z 379.23) and (H2O)22H+ (m/z 397.24) which are known as a magic and an antimagic water cluster number [12]. The intensity differences are in good accordance with the ones found by Conlan et al. [2]. This gap is an important fingerprint in the mass spectrum of water ice and will be emphasized for this measurement in the supplementary material (see figure S10).

Temperature profile for water clusters with different cations. The line color indicates the mass of the cluster ion as shown in the color bar to right of each figure. Signals from the ciprofloxacin overlap with the CO2+ cationized water cluster signals. The temperature of 153 K is indicated by the gray line, the temperature of 163 K by the yellow line

On the same mass at (H2O)n cluster masses, the signals from the NH4+ cationized water cluster ions, the second most intense clusters, show a distinctly different trend (Fig. 2, top right). They rise gradually from 100 to 140 K, drop to a local minimum at 153 K, and then rise to a maximum before the onset of rapid freeze-drying. Eight mass units above the (H2O)n cluster masses, the detected signals in the higher mass range are from CO2+ cationized water clusters (Fig. 2, middle left). In the mass range below m/z 188, CN+ cationized water clusters interfere with CO2+ cationized water clusters. Like the NH4+ cationized water cluster ions, the CO2+ cationized water clusters also show a local minimum at 153 K. This is also true for a total of 9 of the 18 cationized water cluster ion sequences (see figures S6, S7, and S8). The same trend is observed for the total ion yield (not shown). In contrast, the OH+ cationized water clusters, which are the third most intense, go through a maximum at 153 K and then initially decline gradually, then abruptly above 173 K (Fig. 2, middle right).

The ciprofloxacin peaks, which overlap the CO2+ cationized water clusters (Fig. 2, middle left, lines are marked with dots and crosses), are not only much more intense than the CO2+ cationized water clusters, they follow a very different trend with temperature: Ciprofloxacin signals begin to rise around 140 K and continue rising until 173 K and then plummet as the sample rapidly freeze-dries. The water clusters containing CH3+ (Fig. 2, lower left) or CHO+ (Fig. 2, lower right) follow a trend more similar to that of the ciprofloxacin peaks, as do the organic peaks below m/z 100 (data not shown).

Figure 3 shows an overlay of key stable ion peaks vs. temperature for sample 1 normalized to their maximum. The non-protonated ciprofloxacin signal [C17H18FN3O3]+ and the protonated ciprofloxacin signal [C17H18FN3O3+H]+ monotonically increase up to a temperature of 173 K. Above this temperature, rapid freeze-drying occurs and the ciprofloxacin signals decrease rapidly. While the ciprofloxacin signals rise with temperatures, the (H2O)18H+, (H2O)17NH4+, and combined (H2O)nCO2+ (for n = 10, 11, 12, 13, 17, and 18) water cluster signals decline above 140 K. Without risking freeze-drying, an optimum analysis temperature for [ciprofloxacin+H]+ occurs at 153 K with a CO2+ cationized water cluster background reduction of 16–30%. Above the temperature of 153 K, the ciprofloxacin-to-protonated-water-cluster background continues to increase, but the sample begins freeze-drying. The sharp decrease in the ciprofloxacin during rapid freeze-drying suggests that proton transfer from the water ice to the ciprofloxacin enhances the ion yield.

Temperature trends for different cationized water clusters, for protonated and non-protonated ciprofloxacin as well as for NH4+ and Na+ and the total ion signal for Bi3+ analysis of sample 1. (H2O)nCO2+ are combined for the clusters n = 10, 11, 12, 13, 17, and 18 in the mass range of ciprofloxacin, but without interference with ciprofloxacin. The temperature of 153 K is indicated by a gray vertical line, the temperature of 163 K by a yellow vertical line

Similar to protonated ciprofloxacin, the signals of (H2O)nNH4+ and (H2O)nCO2+ increase with increasing temperature above 153 K until rapid freeze-drying occurs. At higher temperatures, freeze-drying results in a sharp rise in NH4+ and elemental signals (such as Na+) which are accompanied by a rise in the signals of the (H2O)nNH4+ clusters. The unusual behavior at 153 K, shortly below the freeze-drying temperature, may be due to surface specific melting. Hong et al. observed significant changes in the mechanical properties of water ice surfaces (between 121 and 152 K) with atomic force microscopy (AFM) measurements which indicated a pre-melting of the ice at 152 K. This surface limited pre-melting could facilitate increased mobility of protons and other charge carriers at the surface of the otherwise frozen, solid ice matter [13]. It is likely that the increased proton mobility in the sample allows competition for positive charge between the solutes at 153 K which leads to the local minima of the signals from (H2O)nNH4+ and (H2O)nCO2+ and the corresponding maximum in the (H2O)nOH+ signal [14, 15].

In order to better understand the unusual behavior of the water signals at 153 K, which is just below the freeze-drying temperature, an additional temperature-controlled ToF-SIMS study was undertaken. A set of measurements was performed on a cell-free model biofilm made with D2O (sample 2).

Figure 4 shows ToF-SIMS spectra from m/z 285 to m/z 375 taken from cell-free model biofilm sample 1 (top), which was made with triply distilled water, and sample 2 (bottom), which was made with D2O and contains a 130:1 ratio of D2O to H2O. In the frozen deuterated sample spectra (bottom), water cluster ion sequences separated by 20.03 u (D2O) are observed. Four repeating patterns can be seen between m/z 288 and m/z 370. From m/z 314 to m/z 336, some ciprofloxacin ions superimpose these heavy water cluster ion sequences. Unlike the light water (H2O) sample (Fig. 4, top), where the protonated clusters are an order of magnitude more intense than the other cationized clusters, the heavy water (D2O) sample (Fig. 4, bottom) shows a repeating pattern of protonated and deuterated water clusters of similar intensity that contain different numbers of protium and deuterium atoms. Protium impure heavy water clusters result from ammonium formate, acetic acid, and ciprofloxacin additives, which have labile hydrogens that can exchange with the initially highly pure D2O solvent. The most intense HxDyO(x+y)/2H/D+ cluster peaks in the spectrum of the heavy water sample (Fig. 4, bottom) are at least a factor of 20 lower intensity than the (H2O)nH+ cluster peaks seen in the spectrum of the light water sample (Fig. 4, top). For larger deuterated water clusters, the statistical probability for protium-containing clusters increases. Accordingly, higher levels of protium ratios are observed in deuterated water clusters for higher mass ranges. In the heavy water (D2O) sample, metastable deuterated water cluster peaks cause strong interference with other cationized deuterated water cluster ions. This prevented further investigation of the cationized water clusters in the deuterated solution (figure S11).

Spectra comparison between hydrated (H2O, top) and deuterated (D2O, bottom) ciprofloxacin solutions. In hydrated solutions, a series of peaks, separated by 18.01 u, is dominating the spectrum. In impure deuterated solutions, deuterated water clusters have incorporated a different amount of hydrogen atoms and form sequences of water cluster peaks separated by 20.03 u

In the heavy water (D2O) sample, ciprofloxacin will readily exchange deuterium for the hydrogen atom attached to the piperazine ring and the hydrogen atom attached to the carboxylic acid group, which leads to three distinct ciprofloxacin molecular ions (see figure S1). Although interference with the metastable background associated with the H5D28O+ cluster ion is significant, the signal for the deuterium substituted ciprofloxacin [C17H16D2FN3O3+D+] is clearly identifiable as well as the ciprofloxacin signals at [C17H16D2FN3O3+H+] and [C17H16D2FN3O3-OD+].

The temperature-dependent intensities of different peaks from a deuterated water cluster sequence with 14 oxygen atoms are shown in Fig. 5. The mass range selected does not include any typical ciprofloxacin fragments. The temperature profiles reveal significantly different temperature trends for clusters depending on the ratio of protium to deuterium. Clusters with a high deuterium-to-protium ratio are most intense at the lowest temperatures. Above 155 K, most water cluster ions observed contain a high protium-to-deuterium ratio. Thus, the exchange of deuterium and protium atoms between D2O and organic molecules is pronounced in this temperature region. Above 158 K, the clusters with a high deuterium-to-protium ratio increase, and the clusters with a low deuterium-to-protium ratio decrease.

Temperature-dependent peak intensities from a D2O water cluster set with 14 oxygen atoms in a mass range without ciprofloxacin fragments. The purest D2O cluster (D2O)14D+ is less intense around 155 K (gray vertical line). The most impure shown water cluster H11D18O14+ is most intense around 155 K. The temperature of 163 K is indicated by a yellow vertical line

It is notable that the water clusters with the smaller deuterium-to-protium ratio (Fig. 5, blue) go through a maximum at 155 K, and the clusters with the larger deuterium-to-protium ratio (Fig. 5, green) go through a minimum at this value. A minimum was also observed in NH4+ cationized water clusters near 153 K in the light water sample (Fig. 2). The slight difference in the optimum temperature between the light and heavy water cell-free biofilm models is consistent with the known differences in phase transition temperatures between light water and heavy water. However, estimating these temperature shifts is challenging, as experimental vapor pressure data in the supercooled regime for D2O are still lacking, even in the most recent data [16].

The signal C17H17D2FN3O3+ (protonated ciprofloxacin with two deuterium substitutions) is most intense at 158 K (Fig. 5, red). This supports the hypothesis of enhanced proton mobility near 155 K. However, interference from the metastable H28D9O18+ peak with the C17H17D2FN3O3+ signal may also be influencing this trend.

For future studies, the role of a variety of different organic compounds on water cluster formation should be investigated. We propose a ciprofloxacin-like temperature behavior, as similar temperature trends were observed for all organic ions in the low mass range in this work. However, Conlan et al. have shown that different analytes and analyte concentrations shift the equilibrium proton binding in the aqueous environment [1]. The influence of sample pH should also be investigated, as pH values affect proton availability and could alter the influence of temperature on the water cluster ion signals.

Hong et al. used an AFM to investigate water ice surface changes during pre-melting [13]. A combined ToF-SIMS and AFM measurement can deliver insights into changes in ion-bombarded ice surfaces, just before freeze-drying occurs.

Structural changes in cryo-samples can lead to changes in the mechanical properties of the ice surface. Poleunis et al. used Ar-backscattering [17] to observe softening of polymer surfaces during a temperature increase. Softer ice could contribute to higher molecular ciprofloxacin signal intensity by altering the nature of the sputter cascade. Further studies using Ar-backscattering could improve understanding of the changes occurring in the ice.