Enhanced Raman multigas sensing – a novel tool for control and analysis of 13 CO 2 labeling experiments in environmental research

Cavity-enhanced Raman multigas spectrometry is introduced as a versatile technique for monitoring of 13 CO 2 isotope labeling experiments, while simultaneously quantifying the ﬂ uxes of O 2 and other relevant gases across a wide range of concentrations. The multigas analysis was performed in a closed cycle; no gas was consumed, and the gas composition was not altered by the measurement. Isotope labeling of plant metabolites via photosynthetic uptake of 13 CO 2 enables the investigation of resource ﬂ ows in plants and is now an important tool in ecophysiological studies. In this experiment the 13 C labeling of monoclonal cuttings of Populus trichocarpa was undertaken. The high time resolution of the online multigas analysis allowed precise control of the pulse labeling and was exploited to calculate the kinetics of photosynthetic 13 CO 2 uptake and to extrapolate the exact value of the 13 CO 2 peak concentration in the labeling chamber. Further, the leaf dark respiration of immature and mature leaves was analyzed. The quanti ﬁ cation of the photosynthetic O 2 production rate as a byproduct of the 13 CO 2 uptake correlated with the amount of available light and the leaf area of the plants in the labeling chamber. The ability to acquire CO 2 and O 2 respiration rates simultaneously also simpli ﬁ es the determination of respiratory quotients (rate of O 2 uptake compared to CO 2 release) and thus indicates the type of combusted substrate. By combining quanti ﬁ cation of respiration quotients with the tracing of 13 C in plants, cavity enhanced Raman spectroscopy adds a valuable new tool for studies of metabolism at the organismal to ecosystem scale.


Introduction
Isotope labeling with gaseous precursors is an important tool in ecophysiological studies as it allows for a detailed investigation of the ow of resources at the level of an individual organism up to an entire ecosystem. For example, by labeling with 13 C, the existence of different carbon allocation patterns between plant functional groups 1 and the fast transfer of recently assimilated carbon to soil microorganisms 2 have been demonstrated. 13 C labeling has also been used at a molecular level to understand plant investments in secondary metabolites that serve as antiherbivore defenses 3 as well as the biosynthetic pathways leading to defenserelated compounds. 4 Chemical ecology studies have monitored photosynthetic uptake of 13 CO 2 in real time in order to measure the incorporation of newly assimilated 13 C into primary versus secondary metabolites under simulated herbivore pressure 4,5 to address the growth-defense hypothesis. 6 Measuring gas exchange of both 13 CO 2 and 12 CO 2 is important for investigations of the balance of the amount of incorporated 13 C versus the amount of respirationally released 13 CO 2 . 13 C labeling of plants via exposure to pulses of 13 CO 2 is becoming a more commonly employed tool in studies of plant physiology and chemical ecology. 7 Nowadays, in most plant respiration experiments, CO 2 production is measured as the sole parameter, either electrochemically or by non-dispersive infrared absorption spectroscopy. 8 However, O 2 consumption is also important for the determination of the respiratory quotient in order to draw conclusions about the type of combusted substrate and for quantication of the amount of label that got xed by the plant. Most commonly used methods are not sufficiently sensitive in the measurement of O 2 in plant respiration over timescales of minutes. High sensitivity O 2 measurements are currently performed by taking gas samples for successive lab-based analyses using gas chromatography (GC) in combination with mass spectrometry (GCMS) in order to determine O 2 /N 2 ratios. Unfortunately, these chromatographic techniques are slow, consumptive, and expensive, because the samples consist of complex mixtures of gases at various concentrations and several expensive test gases are needed for instrument calibration. To date, the miniaturization of test equipment for rapid online monitoring of multigas-samples (consisting of O 2 , N 2 , 13 CO 2 and These limitations can be circumvented by Raman spectroscopy, which provides characteristic information about molecular vibrations 9 and thus chemical specicity. Raman spectroscopy emerged in recent years as an extremely powerful method 10 in various natural science disciplines 11 to investigate solid samples, liquids, and gases. Raman gas analysis is capable of quantifying almost all gases (except noble gases) simultaneously with just one measurement. 12 This work introduces the methodology of cavity enhanced Raman multigas spectroscopy for pulse labeling studies of plant physiology.

Experimental details
Monoclonal cuttings of Populus trichocarpa were obtained from the "Thüringer Landesanstalt für Landwirtscha", Dornburg (Germany). Populus trichocarpa is a fast-growing species native to western North America. Cuttings were individually planted in 2 l pots with potting soil (Klasmann KKS Bio Topfsubstrat 27) mixed 1 : 1 with quartz sand and grown in the greenhouse with additional light (Son-T Agro 430 W HPS bulbs, primary light range 520-610 nm, Philips Lighting Company, New Jersey, USA) from 6:00-17:00. The pots were uniformly watered with an irrigation system that delivered water two to three times for 3 min between 12:00 and 13:00 each day, depending on the temperature. The plants were moved to a climate chamber and exposed to ve days of gradual cooling followed by an articial winter of 8 h 10 C days and 4 C nights to induce senescence and leaf fall. The plants were returned to the greenhouse and exposed to the previous light and water conditions. As leaves were ushing, plants were exposed to 13 CO 2 in a 2 m 3 labeling chamber (see Fig. 2). The gas phase was homogenized with the help of fans. Twenty-three to 25 plants were labeled on two consecutive days (runs 1 and 2, see Table 1) for approximately 2 h from 12:00 to 14:00. 13 CO 2 was introduced into the chamber via acidication of 2.67 g followed by 1.33 g 99% NaH 13 CO 3 (Cambridge Isotope Laboratories, USA) with 16 ml or 8 ml diluted hydrochloric acid. The leaf area of every plant was measured at the time of labeling.

Raman gas monitoring of multigas mixtures containing isotopic labeled gases
The Raman gas sensor consists of a miniaturized laser diode with l exc. ¼ 650 nm and a cw output power of 50 mW. This diode is passively frequency locked and feedback-coupled to a high-nesse cavity (PCB) enabling a power build-up to 100 W. Thus a strong signal enhancement is achieved with only low power consumption of the instrument. The PCB supports a Gaussian beam and consists of an input coupler mirror and an end mirror, both with extremely low scattering losses and transmission. For optimal beam enhancement and stable operation, the cavity components are aligned for spatial mode matching of the input beam and the Gaussian beam supported by the PBC while the facet of the laser diode helps in stabilizing mode matching by spatial ltering. 13 This arrangement of the PCB is extremely stable to mechanical vibrations and is connected to a high-throughput spectrometer with a room temperature operated charge coupled device (CCD) with 512 pixels and a spectral resolution of approx. 50 cm À1 . Additional sensors monitor the laser intensity, pressure and temperature for reliable gas quantication. With the help of this strong signal enhancement it was possible to monitor concentration uctuations of about 50 to 100 ppm within measurement times of one second. The device was calibrated for the relevant gases, N 2 , O 2 , 12 CO 2 , and 13 CO 2 , by ushing the optical cavity with pure gases. Underground correction was accomplished by subtracting the spectrum of the Raman inactive noble gas argon. 13 CO 2 was calibrated with a GCMS-validated 1% mixture of 13 CO 2 with 99% argon (Raman inactive noble gas). The calibrated reference spectra are a prerequisite for the online quantication of sample gases during the isotope labeling experiment (Fig. 1). A straightforward calibration approach is feasible because the Raman intensity, I Stokes , scales strictly linearly with the gas density in molecules per volume, nV À1 , laser power, I 0 , and partial pressure, p over the whole dynamic range.
Next, a least square t of the measured spectrum and the calibrated reference spectra provided the concentrations of the individual gases. Therefore an over-determined linear equation system was solved with the calibration gas, g, measured gas, a (mixture of gases), intensity, I(ñ), concentration, c, CCD pixel, n, and number, m, of extracted gases. (2) Another major advantage of Raman multigas sensing becomes obvious from eqn (2), namely that all gases which Table 1 Comparison of the Raman gas monitoring of two labeling runs with different leaf areas and available photosynthetically active radiation (PAR) in the labeling chamber. The amount of developed oxygen, mean PAR, total leaf area and the exponential decay time t e are given. The oxygen production rate is higher and the CO 2 uptake is faster with higher light intensity and larger leaf area in labeling run 2. t e represents the decay time until the concentration decreased to 1/e of its initial value based on the exponential fit equation: appear in course of a labeling experiment will be detected in the Raman spectrum of the multigas-mixture. Thus, if the difference between the experimental multigas-spectrum and the deconvoluted individual spectra differs from a zero-baseline, more information about additional gases can be obtained by data post-processing with an increased number, m, of extracted gases. An example spectrum of an experimental gas mixture and the spectra of the gaseous components (N 2 , O 2 , and CO 2 ) are depicted in Fig. 1A. The Raman gas spectra of the chemically similar gaseous isotopes, 12 CO 2 and 13 CO 2 , can be readily distinguished due to their spectral shi and differences in the intensity pattern of the Fermi diad 14 (Fig. 1B). Thus, all relevant gases (N 2 , O 2 , 12 CO 2 , and 13 CO 2 ) can be quantied individually and simultaneously with no cross-sensitivity. The gas concentrations (N 2 , O 2 , 12 CO 2 , 13 CO 2 ) obtained were normalized for a constant sum of all gases and a baseline subtraction was done for 12 CO 2 and 13 CO 2 .

CO 2 labeling experiment of Populus trichocarpa
The utility of the new multigas sensing methodology is demonstrated in a labeling experiment that was designed to investigate the allocation of newly assimilated carbon to secondary metabolites. Saplings of Populus trichocarpa (see Experimental details) were exposed to 13 CO 2 in the chamber during a two hour pulse labeling experiment (Fig. 2). The Raman gas sensor was connected to the labeling chamber on the opposite side of the 13 CO 2 input to measure the labeled gas aer it traveled through the chamber (Fig. 2). The Raman sensor always analyzed the homogenized gas concentration of the labeling chamber. Successive addition of the label was applied to increase the levels of 13 C that could be incorporated into plant metabolites. Raman gas monitoring was applied to observe the maximum 13 CO 2 concentration and to ensure that the plants took up all the labeled 13 C. The continuous online quantication of the 13 CO 2 level in the chamber allowed for accurate monitoring of the uptake during the labeling period and the precise timing of the second dose of 13 CO 2 (Fig. 3). First, the labeling chamber was ushed with CO 2 -free air to decrease the amount of 12 CO 2 from 586 ppm to <100 ppm within 0.5 h. At 0.7 h, 13 CO 2 was chemically generated from 13 C-bicarbonate and diluted hydrochloric acid. A few minutes later the concentration of 13 CO 2 reached its maximum at 298 ppm and then decreased to <100 ppm aer 1.6 h due to Fig. 1 (A) Example of a Raman gas spectrum (l exc. ¼ 650 nm) during a typical leaf dark respiration measurement of Populus trichocarpa. The Raman spectrum of the unknown gas mixture (grey) and the Raman spectra of the individual gaseous components (green: N 2 , blue O 2 , black CO 2 ) are shown (N 2 and O 2 are ro-vibrational spectra, with unresolved O and S branches). The concentrations of the individual gases can be deconvoluted from the experimentally acquired envelope. (B) The Raman gas spectra of 12 CO 2 and 13 CO 2 can be distinguished and simultaneously quantified due to their spectral shift and differences in the intensity distribution of the Fermi diad. Fig. 2 Schematic setup of the Raman gas analysis of a 13 CO 2 labeling experiment. The Raman gas sensor was connected to the labeling chamber such that the homogenized gas of the chamber was analyzed in a closed cycle. The Raman analysis did not change the gas composition. 13 CO 2 was released chemically and introduced into the chamber by a valve. The inset-photograph of the labeling chamber shows the seedlings of Populus trichocarpa under growing lights in order to stimulate photosynthetic uptake of 13 CO 2 . photosynthetic uptake by the plants (Fig. 3). Aer the complete uptake of the rst dose, a second dose was administered. The 13 CO 2 concentration reached 182 ppm at 1.8 h and dropped to <100 ppm during the next half hour. At 2.75 h the labeling chamber was opened to ambient air. The concentration of O 2 (calculated by a linear t) rose by approximately 400 ppm during the labeling period, whereas the concentration of N 2 did not change signicantly. A major drawback of conventional gas sampling techniques is the extended time needed for data analysis and consequently the small number of data points. In contrast, the high time resolution of Raman gas sensing was exploited for the rapid acquisition of many data points tracking the 13 CO 2 -concentration during the course of the labeling, which allowed for kinetic investigations. First-order exponential tting enabled the very precise determination of time constants (Table 1) and peak concentrations of 13 CO 2 ( Table 2).
Two separate labeling runs were performed on different days, and more leaf area and photosynthetically active radiation (PAR) were available inside the labeling chamber in the second run. The comparison of both runs revealed that the amount of O 2 produced during the 13 CO 2 labeling period was higher (0.38 mol per day compared to 0.35 mol per day) in run two. Similarly, the uptake of 13 CO 2 over time was faster in the second run, with a decay time of 15.7 min to reduce the amount of 13 CO 2 to 1/e of its starting value in comparison to 20.6 min in the rst labeling run. All values are summarized in Table 1.
The high time resolution of Raman gas monitoring (Fig. 3) enabled the detection of small deviations from the exponential decay due to uctuations in natural light intensity, and, by comparison of both doses, it was even possible to conrm that 13 CO 2 uptake by P. trichocarpa was faster at higher concentrations of 13 CO 2 within the course of the rst dose because the photosynthesis rate of C 3 plants is not strictly linear at low concentrations. 15 An important task in environmental labeling experiments is the correct estimation of the peak concentration of 13 CO 2 in the labeling chamber. Peak concentrations are conventionally calculated based on the mass of the 13 C-bicarbonate used to create the 13 CO 2 . However, these approximations overestimate the value in the homogenized chamber atmosphere, due to the immediate photosynthetic 13 CO 2 -uptake by the plants in the labeling chamber and the time for the chemical release of 13 CO 2 which broadens the sharpness of the labeling pulse. It is more precise to measure the 13 CO 2 -concentration in the labeling chamber online and with rapid data acquisition by means of Raman gas sensing and calculate the peak concentration from the decay equation with high precision by extrapolating back to the time of the dose ( Table 2). These peak concentrations of 13 CO 2 were lower with the rst addition of 13 CO 2 than those calculated based on the mass of reacted bicarbonate, but the extrapolated values were higher aer the second addition of 13 CO 2 in each labeling run (Table 2). This demonstrates that residual 13 CO 2 from the rst dose was still present at the addition of the second dose, meaning actual 13 CO 2 values were noticeably higher than expected by standard bicarbonateweight based calculations ( Table 2).
In general, the amount of available data points from the temporally highly resolved Raman spectroscopic gas measurements enabled a very reliable tting of time dependency curvatures and was well suited for kinetic investigations.

Leaf dark respiration
The labeled plants were further investigated with leaf dark respiration measurements in order to understand 13 CO 2 exchange over time. Approximately 24 h aer the 13 C-labeling, P. trichocarpa leaves of a known area were enclosed in a dark chamber, and the concentrations of O 2 , 12 CO 2 , and 13 CO 2 were continuously recorded. The uxes of all gases were calculated based on changes of the gas concentrations over time in the enclosed headspace. The uxes were related to a leaf area that was based on one side of the leaf only to represent the area where the stomata are located. The respired gases were circulated through the instrument and returned to the chamber without consumption or alteration by the measurement. Ambient air was measured aer one hour to validate the stability of experimental setting. A linear t of the gas concentrations over time yielded rates for the uptake of O 2 and CO 2 production (Fig. 4). The current detection limit of the device for Table 2 Summary of the 13 CO 2 peak concentrations (ppm) released. The calculated values were derived from the weighted 13 C-bicarbonate portion. The extrapolated values were derived from the exponential fit of the measured 13 CO 2 Raman gas curve. Both calculations are in agreement, however the extrapolated Raman gas data deliver more precise information of the homogenized chamber gas atmosphere due to the fast photosynthetic 13 CO 2 -uptake during the time of the chemical 13  These results indicate that the respiration rates of immature leaves are more than twice as high as the respiration rates of mature leaves. Distinctly higher dark respiration rates of immature leaves are also reported in the literature. 16 Simultaneous Raman spectroscopic quantication of O 2 and CO 2 also allowed for the calculation of the respiratory quotient (RQ) which yields information about the compounds being metabolized and respired. Deviations in the respiratory quotient arise from differing carbon-oxygen ratios of substrates or the formation of byproducts. 17 The RQ values were $1 for all leaves, indicating the combustion of starch. 18

Conclusions and outlook
This work demonstrates the unique capabilities of innovative cavity enhanced Raman gas monitoring for the control and analysis of 13 CO 2 -labeling experiments. Enhanced Raman gas sensing is superior to conventional Raman gas spectroscopy, due to the strong power build up to 100 W within the cavity (while maintaining low instrument power consumption) and outperforms other gas sensing techniques for the rapid and simultaneous analysis of multiple gases in a labeling chamber while eliminating sample collections and delayed analyses. The technique is non-consumptive, such that the measurements can be performed in a closed cycle with the labeling chamber without altering the gas composition. The high time resolution of the Raman measurement enables the acquisition of a huge number of data points, which tremendously increases the accuracy of kinetic investigations. Thus, it was possible to determine precise uptake rates and peak concentrations of 13 CO 2 in the pulse labeling of P. trichocarpa. Additionally, the simultaneous measurements of CO 2 and O 2 allowed for calculation of photosynthetic rates for both gases at once which correlated with the leaf area as well as the photosynthetically active radiation inside the labeling chamber. The investigation of leaf dark respiration of P. trichocarpa revealed that the respiration rate of immature leaves was more than twice as high compared to mature leaves. Simultaneous Raman gas quanti-cation of O 2 and CO 2 enabled the calculation of the respiratory quotient, which is an indicator of the chemistry of metabolites that are fueling respiration, or can indicate the net effects of gas transport via the plant transpiration stream. Monitoring of 13 CO 2 , 12 CO 2 , and O 2 also allows for quantication of the amount of label that got xed by the plant and the 13 C : 12 Cratio. Cavity enhanced Raman multigas sensing was shown to be a very versatile new technique for monitoring the amount of label incorporation in 13 CO 2 -labeling experiments and it is also capable of rapidly analyzing the respiration quotient, an ecophysiologically important parameter. This new technique is affordable and very robust due to the linearity of signal intensity to analyte concentration. We therefore anticipate that cavity enhanced Raman spectroscopy (CERS) will become an important tool for labeling experiments in environmental research.