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Measurement and evaluation of FT-Raman spectra of Norway spruce needles: how the background variability can be explained
Journal of Molecular Structure 661-662 (2003) 333–345 www.elsevier.com/locate/molstruc
Measurement and evaluation of FT-Raman spectra of Norway spruce needles: how the background variability can be explained P. Matejka*, H. Tokarova´, T. Pekarek, K. Volka Department of Analytical Chemistry, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28 Czech Republic Received 28 April 2003; revised 4 June 2003; accepted 2 July 2003 To Professor Dr Bernhard Schrader, in whose laboratory I (K.V.) measured my first FT-Raman spectrum and experienced his generous hospitality and friendship
Abstract A series of model experiments both in vitro and in vivo were aimed to describe the effects of variability of the background shape underlying the FT-Raman spectral features of Norway spruce needles. Detailed FT-Raman spectral measurements of individual needles, of separated twigs and of small living trees were evaluated on the basis of calculation of statistical spectra and using principal component analysis. The increase of the baseline slope was ascribed to the effect of drying out, while the broad emission background is related to local impurities/injuries on the needle surface. q 2003 Elsevier B.V. All rights reserved. Keywords: Biospectroscopy; Picea abies; In vitro; In vivo; Chemometric evaluation
1. Introduction Plant needles have been often utilized as a bioindicator of environmental pollution. They serve not only as natural samplers of air pollutants but the changes of their surface micromorphology and of the chemical composition can be correlated with an environmental stress of the vegetation. Norway spruce is a typical tree of middle Europe; therefore, the Norway spruce needles are well suited for environmental monitoring in this area. An analysis of Norway spruce needles (Picea abies (L.) Karst.) by FT-Raman spectroscopy [1 – 3] and ATR technique in mid* Corresponding author. Tel.: þ420-2-2435-4281; fax: þ 420-22431-0352. E-mail addresses: [email protected] (P. Matejka); karel. [email protected] (K. Volka). 0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-2860(03)00445-9
infrared range [4] has been already reported. Schrader et al. [5] has described that living cells are possible to measure by FT-Raman spectroscopy using a nearinfrared (NIR) laser source with only small contribution (or none at all) of fluorescence and with only a small risk of damage to living cells. Schrader et al. [6] expressed the conditions of non-destructive analysis that have to be fulfilled in the case of Raman biospectroscopy. The main problem of bioindicator analysis is to distinguish the natural factors, the anthropogenic influences and the effects of the analysis itself. To explain the data variability observed by the FT-Raman spectroscopic analysis of needles taken from a large set of trees from six different forest areas in various seasons, we have performed a series of model experiments in vitro on cut-off twigs and individual needles and, furthermore,
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in vivo on small trees which were placed directly in the sample compartment of the FT-Raman instrument. The living trees, the separated twigs and the individual needles were fixed for a few weeks in the spectrometer. The effects of dark periods and of artificial lighting were studied in all cases. Detailed studies of multiple points on some individual needles were aimed to distinguished the general characteristics of needles and the local effects. This study is focused on the evaluation of the background shape underlying the Raman bands in the FT-Raman spectra measured, because roughly 10% of needles of large data sets (thousands of spectra measured in last five years) collected for environmental monitoring exhibited intense unusual background shape, that can be characterized either by a sloping baseline or by a broad band emission. The main challenge is to get relevant information not only from Raman bands but also from the shape of the background. 2. Experimental 2.1. Preparation and treatment of trees, twigs and needles for analysis The small trees have to be carefully prepared to live for a few weeks in the compartment of the spectrometer (Fig. 1(A)). The root ball was protected against desiccation of soil and its overheating in the lighting periods by fixing the plant pot in a beaker that was wrapped on sides and bottom in Al foil and covered from the top side by Parafilm ‘M’ foil. The root ball was fastened to the sample stage in the sample compartment of the spectrometer using doublesided adhesive tape. A polymethymethacrylate ring with a laterally bored hole and a cavity on the opposite side was used for stable fixation of a selected needle in the measuring position. The transparent ring enabled also irradiation of the needle by a halogen lamp during the model experiments in which artificial lighting on the tree and dark periods were undertaken. The same ring was also used to fix individual needles, which were carefully torn off branches using tweezers, and needles measured on cut-off twigs. Two of the selected twigs were prevented against drying within the two-week experiment. These twigs were placed through a bored cap in a polypropylene tube filled with cotton-wool soaked with fresh drinking
water to ensure the water supply (Fig. 1(B)). The tube with the cap was packed using Parafilm ‘M’ and then it was covered by Al foil to prevent overheating during lighting periods. 2.2. Instrumentation 2.2.1. FT-Raman spectroscopy FT-Raman spectra were collected using a Fourier transform near-infrared (FT – NIR) spectrometer Equinox 55/S with FT-Raman module FRA 106/S (Bruker). The fixed needles were irradiated by the focused laser beam with a laser power 50 mW of Nd:YAG laser (1064 nm, Coherent). The scattered light was collected in backscattering geometry. A quartz beamsplitter and Ge detector (liquid N2 cooled) were used to obtain inteferograms. 1024 scans were accumulated for an individual spectrum. A standard 4 cm21 spectral resolution, ‘zero filling’ 8 and Blackmann– Harris cosine apodization function was used for all data accumulation and Fourier transform processing. 2.2.2. Optical microscopy To check the surface of the needles the microimages from optical microscope were recorded. The trinocular microscope Optiphot 2 (Nikon) with objectives 10 £ , 20 £ , 40 £ and 100 £ (Nikon) was equipped with a color CCD camera (Sony). The camera was connected directly to PCI TV card (TV Capturer) inserted in standard Pentium computer. The software TVIEW 98 was used to obtain, to process and to save microimages. 2.3. Scheme of experiments 2.3.1. Detailed analysis of individual needles Eight needles, which exhibited unusual background shape in the case of standard experiments performed for environmental monitoring in the year 2002, were selected for detailed analysis in multiple points. Each needle was placed vertically in the sample holder with the tip oriented downwards. The first spectrum of a needle was collected in its central part. Then the needle was moved up using the step-wise moving sample stage. The new site was about 1 mm closer to the tip with respect to the initial position. The next position was 1 mm in the opposite direction from the initial position. The needle was then moved up about 3 mm to
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Fig. 1. Experimental arrangement in the sample compartment of the spectrometer: (A) a living tree fixed in the sample compartment, (B) a twig supplied with water.
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obtain the fourth spectrum at the distance of 2 mm from the initial position to the tip. The final spectrum was recorded in the position 2 mm from the initial one to the base of the needle. In all, we obtained five spectra from five different places on one side of the needle, where the distance between the endpoints was 4 mm. The same experiment was repeated on the opposite side of the needle. Summarily, we recorded ten spectra per needle. 2.3.2. Time-dependent experiments Some general conditions were kept during all the time-dependent experiments to ensure compatibility of data obtained. Eight spectra were recorded consecutively during every day of measurement to allow evaluation of the daily data variability using averaged data and standard deviation records. The needle examined was not moved during the two-week experiment to exclude the variability of data that can be caused by differences expected from different parts of the needle. Analysis of needles on living trees. The experiments were performed on two different small trees. The time-dependent experiments were carried out on two needles from different twigs for each of the trees, which means that four sets of experiments were evaluated. The night dark periods and artificial daylight periods were regularly alternated. Analysis of needles on cut-off twigs. Four cut-off twigs were examined. Two of them were supplied with water to protect them against drying (as described above). The other two were left under ambient laboratory conditions with exception that all four twigs were exposed to the regularly alternated dark periods and artificial light periods. Analysis of individually separated needles. Three individual needles were studied in the time-dependent experiments. Two of them were exposed to the dark and light periods as described above for the living trees and cut-off twigs. The last needle was kept in the dark during the two-week experiment to eliminate the effect of artificial lighting. 2.4. Treatment and evaluation of spectra All treatment of spectra was undertaken using the software OPUS NT 4.0 (Bruker). Eight measured spectra were primarily averaged for each of date of
measurement in the case of the time-dependent experiments. The standard deviation curve was also calculated. The averaged spectra and the standard deviation curves were also calculated for needles measured in 10 different points. Every measured and calculated spectrum was cut to the range 3600 – 90 cm21 and exported to JCAMP-DX format. The JCAMP-DX data were imported to The Unscrambler v. 7.8 (Camo, Norway) for the chemometric evaluation. The principal component analysis (PCA) of various data sets was used to obtain the data distribution (‘scores’) along principal components (PCs) and to compare the x-loadings of individual PCs with the measured spectra.
3. Results and discussion 3.1. Analysis of FT-Raman spectra obtained on multiple points of needles The ten FT-Raman spectra obtained for each needle were mutually compared and analyzed using PCA. This comparison was made for each of eight separated needles examined. The sloping baseline shape, especially in the low frequency range (below ca. 1000 cm21), is observed for all the data obtained on the particular needle (for example Fig. 2(A)), while the broad band emission is observed only on one or two points on the needle examined (e.g. Fig. 2(B)). In the case of Raman spectra of needles characterized by a sloping baseline, the scores plots over the first PC (PC1) and the second PC (PC2) as a result of the PCA analysis demonstrate that the sign of the PC1 is related to the side of the needle which the data were collected from (Fig. 3(A)). No outliers were observed in these cases. The xloadings curve of PC1 (Fig. 3(B)) does not show any Raman spectral band, the shape corresponds to the effect of various offsets of the spectra, especially in the range below 3000 cm21. The sloping baseline observed in the spectra below ca. 1000 cm21 is not displayed in the x-loadings of PC1, because the slope is similar in all the spectra recorded. The graph of x-loadings of PC2, which explains only 4% of the variance compared with 93% attributed to PC1, exhibits a contribution of some Raman bands
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Fig. 2. FT-Raman spectra obtained in different places on a needle: (A) ten spectra of a needle I (sloped baseline), (B) ten spectra of a needle II (broad band emission in one place, usual background in the other places). The spectra measured on side 1 of a needle are dotted; the spectra measured on side 2 are full lines.
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Fig. 3. The results of PCA for Raman spectra with sloped baseline measured on one needle in ten different points: (A) scores plot, (B) x-loadings of PC1, (C) x-loadings of PC2. Individual points of scores plot are marked by a code x M, where x ¼ 1; 2 is the side of a needle, M ¼ A, B, C, D, E are the points along the needle on the specified side.
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Fig. 4. The results of PCA for ten Raman spectra of a needle, where a spectrum with broad band emission was measured: (A) scores plot, (B) xloadings of PC1, (C) x-loadings of PC2 Individual points of scores plot are marked by a code x M, where x ¼ 1; 2 is the side of a needle, M ¼ A, B, C, D, E are the points along the needle on the specified side.
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and a weak negative contribution of the sloping baseline below 900 cm21 (Fig. 3(C)). Different scores plots are observed in the case of a needle for which one or two spectra exhibited broad band emission. As shown in Fig. 4(A), such spectra are on the scores plots represented by outlying points. Clustering of the other data is based on the side of the needle, which the spectra were collected from. The shape of the x-loadings of PC1 (Fig. 4(B)) of course corresponds to the difference of the outlier from the other points, i.e. a broad band emission feature is observed. The graph of x-loadings of PC2, which explains only 1% of the variance compared with 99% attributed to PC1, shows a contribution of some Raman bands to the data variability (Fig. 4(C)). This weak effect of Raman spectral bands to the second principal component is the only one evident feature analogous to the PCA results of the data characterized by the sloping baseline. The surface of needles was checked using optical microscopy. The healthy stomatal antechambers
covered by a porous waxy layer can be easily distinguished because the diffuse reflection of white light is observed on these places. No anomalous local effects were observed on the needles, for which the spectra with sloping baseline were collected. On the other hand, abnormal features were observed in the areas (less than 0.8 mm2) of needles, where the spectra with the intense broad emission were recorded. The reflecting waxy layer was damaged, or even it was missing, extraneous black objects were observed on the needle surface, sometimes overlapping the area of the stomatal antechamber (e.g. Fig. 5). In conclusion, the sloping baseline of the FTRaman spectra is an overall feature of some needles. No abnormal effects are observed on the surface of these needles using optical microscopy. The broad emission background is specific for FT-Raman spectra recorded from local areas on the needle surface. The defects on the waxy layer covering stomatal antechambers and/or extraneous impurities are observed in these surface areas.
Fig. 5. Microimage of a needle in the area, which the spectrum with broad band emission was taken from. The stomatal antechambers are marked with circles (diameter of circles ca. 70 mm). The mostly damaged stomatal antechamber is highlighted by an arrow.
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Fig. 6. Averaged FT-Raman spectra of a needle for selected days during the time-dependent experiment performed on an individual needle: (A) day 1, (B) day 3, (C) day 4. (D) day 8, (E) day 14.
3.2. Analysis of FT-Raman spectra of time-dependent experiments Another viewpoint on the spectra with sloping baseline can be based on the results of model
time-dependent experiments performed on individual needles, cut-off twigs and small living trees. A quite significant increase in the slope of the spectral baseline during the two weeks of the experiment was observed for all the separated
Fig. 7. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a cut-off twig kept without external water supply: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
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Fig. 8. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a cut-off twig kept with external water supply: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
needles examined regardless of the lighting schedule (e.g. Fig. 6). The increase of the baseline slope proceeds within the whole duration the experiment. It should be noticed that a decrease of a weak broad band at 3220 cm 21, attributable to a water vibrational mode, can be reliably observed in the first few days.
A slower and weaker increase of the baseline slope was observed in the FT-Raman spectra of needles on the cut-off twigs, which were kept without a water supply (Fig. 7), in comparison with the data from the separated needles. No changes of the spectral background are characteristic for the spectra collected for the needles on the cut-off twigs supplied with water (Fig. 8).
Fig. 9. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a living tree: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
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At the same time, the intensity of the band at 3220 cm21 is apparently stable during the two-week experiments. Similar effects are observed for the data collected on living trees (Fig. 9). There are no evident changes of the background shape of the spectra during
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the two weeks and also there are no significant changes of the band at 3220 cm21 attributed to water stretching vibrational modes. The trends of the changes in spectral baseline were confirmed on the basis of evaluation of
Fig. 10. Plot of x-loadings for data of a time-dependent experiment on a twig kept without water supply: (A) graph for PC1, (B) graph for PC2. (80 FT-Raman spectra evaluated).
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x-loadings as PCA results, both in the case of experiments on individual separated needles and in the case of twigs kept without a water supply (Fig. 10). The plots of x-loadings of PC1 dominated by the sloped baseline demonstrate the changes of
the baseline slope during the two-week experiments. The variability of Raman bands is then mainly observed on the x-loadings of PC2, but the contribution of PC2 to the overall explained variance is quite minor (5%).
Fig. 11. Plot x-loadings for data of a time-dependent experiment on a twig supplied with water: (A) graph for PC1, (B) graph for PC2. (80 FTRaman spectra evaluated).
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The stability of the baseline noted in the case of twigs with a water supply and in the experiments on living trees was proven on the basis of x-loadings plots for PC1 and PC2. Only Raman spectral bands contribute to x-loadings of PC1 and PC2 for the data collected both on living trees and on twigs supplied with water (Fig. 11). Hence, the water supply eliminates the increase of baseline slope during the time-dependent experiments. Summarizing the data of these time-dependent experiments we can conclude that the increase of the baseline slope is related to the desiccation of the needles. The separated needles become dry faster than the needles on cut-off twigs. The desiccation process is eliminated on living trees or on twigs supplied with water. The moisture of a needle is of course its general characteristics and so its does not significantly depend on its examined part, as was shown in the study of the FT-Raman spectra collected in multiple points on individual needles. The baseline shape is much more sensitive to the moisture of the needle than the intensity of quite weak broad band at 3220 cm21 attributed to water stretching vibrational modes.
4. Conclusions The increase of the background slope was attributed to the effect of drying process of the whole needle. The broad band emission underlying the Raman spectral bands is not a characteristic feature of the whole needles, but it can be ascribed to local defects or impurities on the surface of a needle. The results obtained are important from several points of view. Firstly, it is important to improve the methodology of measurements of standard experiments for environmental monitoring. If the broad band emission is observed, other parts of the needle have to be examined to determine the local source of this effect. On the other hand, if the spectrum with sloping baseline is obtained; there is no reason to perform measurements in multiple points to confirm this shape of the baseline. Secondly, if a spectrum with sloping baseline is recorded, the origin of
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the desiccation has to be found. Hence, the conditions of needle storage have to be checked to eliminate the risk of needle desiccation. Thirdly, the abundance of spectra with either sloping baseline or broad band emission is not uniform for all the forest areas monitored. Hence, in the case of appropriate storage conditions, the shapes of the baseline and the abundance of occurrences of such effects have to be considered in the data evaluation, because these observations are related either to a fraction of dry (dead) needles of the specified age at the locality or to the degree of contamination and/or damage of the surface of needles. Of course, the variation of the baseline shape is not the only effect observed in these experiments. The evaluation of the variability of some Raman bands and its consequences will be the topic of a forthcoming study [7].
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant SI 340/1/01) and of the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 223400008) is gratefully acknowledged.
References [1] J. Krizova, P. Matejka, G. Budinova, K. Volka, J. Mol. Struct. 480– 481 (1999) 547. [2] P. Matejka, L. Pleserova, G. Budinova, K. Havirova, J. Nahlik, F. Skacel, K. Volka, Proc. SPIE-Int. Soc. Opt. Eng. 4199 (2001) 130. [3] P. Matejka, L. Pleserova, G. Budinova, K. Havirova, X. Mulet, F. Skacel, K. Volka, J. Mol. Struct. 565–566 (2001) 305. [4] L. Pleserova, G. Budinova, K. Havirova, P. Matejka, F. Skacel, K. Volka, J. Mol. Struct. 565–566 (2001) 311. [5] B. Schrader, H.H. Klump, K. Schenzel, H. Schulz, J. Mol. Struct. 509 (1999) 201. [6] B. Schrader, B. Dippel, I. Erb, S. Keller, T. Lo¨chte, H. Schulz, E. Tatsch, S. Wessel, J. Mol. Struct. 480–481 (1999) 21. [7] P.Matejka, H. Tokarova, T. Pekarek, K. Volka, in preparation.
Measurement and evaluation of FT-Raman spectra of Norway spruce needles: how the background variability can be explained P. Matejka*, H. Tokarova´, T. Pekarek, K. Volka Department of Analytical Chemistry, Institute of Chemical Technology, Technicka 5, Prague 6, CZ 166 28 Czech Republic Received 28 April 2003; revised 4 June 2003; accepted 2 July 2003 To Professor Dr Bernhard Schrader, in whose laboratory I (K.V.) measured my first FT-Raman spectrum and experienced his generous hospitality and friendship
Abstract A series of model experiments both in vitro and in vivo were aimed to describe the effects of variability of the background shape underlying the FT-Raman spectral features of Norway spruce needles. Detailed FT-Raman spectral measurements of individual needles, of separated twigs and of small living trees were evaluated on the basis of calculation of statistical spectra and using principal component analysis. The increase of the baseline slope was ascribed to the effect of drying out, while the broad emission background is related to local impurities/injuries on the needle surface. q 2003 Elsevier B.V. All rights reserved. Keywords: Biospectroscopy; Picea abies; In vitro; In vivo; Chemometric evaluation
1. Introduction Plant needles have been often utilized as a bioindicator of environmental pollution. They serve not only as natural samplers of air pollutants but the changes of their surface micromorphology and of the chemical composition can be correlated with an environmental stress of the vegetation. Norway spruce is a typical tree of middle Europe; therefore, the Norway spruce needles are well suited for environmental monitoring in this area. An analysis of Norway spruce needles (Picea abies (L.) Karst.) by FT-Raman spectroscopy [1 – 3] and ATR technique in mid* Corresponding author. Tel.: þ420-2-2435-4281; fax: þ 420-22431-0352. E-mail addresses: [email protected] (P. Matejka); karel. [email protected] (K. Volka). 0022-2860/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-2860(03)00445-9
infrared range [4] has been already reported. Schrader et al. [5] has described that living cells are possible to measure by FT-Raman spectroscopy using a nearinfrared (NIR) laser source with only small contribution (or none at all) of fluorescence and with only a small risk of damage to living cells. Schrader et al. [6] expressed the conditions of non-destructive analysis that have to be fulfilled in the case of Raman biospectroscopy. The main problem of bioindicator analysis is to distinguish the natural factors, the anthropogenic influences and the effects of the analysis itself. To explain the data variability observed by the FT-Raman spectroscopic analysis of needles taken from a large set of trees from six different forest areas in various seasons, we have performed a series of model experiments in vitro on cut-off twigs and individual needles and, furthermore,
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in vivo on small trees which were placed directly in the sample compartment of the FT-Raman instrument. The living trees, the separated twigs and the individual needles were fixed for a few weeks in the spectrometer. The effects of dark periods and of artificial lighting were studied in all cases. Detailed studies of multiple points on some individual needles were aimed to distinguished the general characteristics of needles and the local effects. This study is focused on the evaluation of the background shape underlying the Raman bands in the FT-Raman spectra measured, because roughly 10% of needles of large data sets (thousands of spectra measured in last five years) collected for environmental monitoring exhibited intense unusual background shape, that can be characterized either by a sloping baseline or by a broad band emission. The main challenge is to get relevant information not only from Raman bands but also from the shape of the background. 2. Experimental 2.1. Preparation and treatment of trees, twigs and needles for analysis The small trees have to be carefully prepared to live for a few weeks in the compartment of the spectrometer (Fig. 1(A)). The root ball was protected against desiccation of soil and its overheating in the lighting periods by fixing the plant pot in a beaker that was wrapped on sides and bottom in Al foil and covered from the top side by Parafilm ‘M’ foil. The root ball was fastened to the sample stage in the sample compartment of the spectrometer using doublesided adhesive tape. A polymethymethacrylate ring with a laterally bored hole and a cavity on the opposite side was used for stable fixation of a selected needle in the measuring position. The transparent ring enabled also irradiation of the needle by a halogen lamp during the model experiments in which artificial lighting on the tree and dark periods were undertaken. The same ring was also used to fix individual needles, which were carefully torn off branches using tweezers, and needles measured on cut-off twigs. Two of the selected twigs were prevented against drying within the two-week experiment. These twigs were placed through a bored cap in a polypropylene tube filled with cotton-wool soaked with fresh drinking
water to ensure the water supply (Fig. 1(B)). The tube with the cap was packed using Parafilm ‘M’ and then it was covered by Al foil to prevent overheating during lighting periods. 2.2. Instrumentation 2.2.1. FT-Raman spectroscopy FT-Raman spectra were collected using a Fourier transform near-infrared (FT – NIR) spectrometer Equinox 55/S with FT-Raman module FRA 106/S (Bruker). The fixed needles were irradiated by the focused laser beam with a laser power 50 mW of Nd:YAG laser (1064 nm, Coherent). The scattered light was collected in backscattering geometry. A quartz beamsplitter and Ge detector (liquid N2 cooled) were used to obtain inteferograms. 1024 scans were accumulated for an individual spectrum. A standard 4 cm21 spectral resolution, ‘zero filling’ 8 and Blackmann– Harris cosine apodization function was used for all data accumulation and Fourier transform processing. 2.2.2. Optical microscopy To check the surface of the needles the microimages from optical microscope were recorded. The trinocular microscope Optiphot 2 (Nikon) with objectives 10 £ , 20 £ , 40 £ and 100 £ (Nikon) was equipped with a color CCD camera (Sony). The camera was connected directly to PCI TV card (TV Capturer) inserted in standard Pentium computer. The software TVIEW 98 was used to obtain, to process and to save microimages. 2.3. Scheme of experiments 2.3.1. Detailed analysis of individual needles Eight needles, which exhibited unusual background shape in the case of standard experiments performed for environmental monitoring in the year 2002, were selected for detailed analysis in multiple points. Each needle was placed vertically in the sample holder with the tip oriented downwards. The first spectrum of a needle was collected in its central part. Then the needle was moved up using the step-wise moving sample stage. The new site was about 1 mm closer to the tip with respect to the initial position. The next position was 1 mm in the opposite direction from the initial position. The needle was then moved up about 3 mm to
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Fig. 1. Experimental arrangement in the sample compartment of the spectrometer: (A) a living tree fixed in the sample compartment, (B) a twig supplied with water.
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obtain the fourth spectrum at the distance of 2 mm from the initial position to the tip. The final spectrum was recorded in the position 2 mm from the initial one to the base of the needle. In all, we obtained five spectra from five different places on one side of the needle, where the distance between the endpoints was 4 mm. The same experiment was repeated on the opposite side of the needle. Summarily, we recorded ten spectra per needle. 2.3.2. Time-dependent experiments Some general conditions were kept during all the time-dependent experiments to ensure compatibility of data obtained. Eight spectra were recorded consecutively during every day of measurement to allow evaluation of the daily data variability using averaged data and standard deviation records. The needle examined was not moved during the two-week experiment to exclude the variability of data that can be caused by differences expected from different parts of the needle. Analysis of needles on living trees. The experiments were performed on two different small trees. The time-dependent experiments were carried out on two needles from different twigs for each of the trees, which means that four sets of experiments were evaluated. The night dark periods and artificial daylight periods were regularly alternated. Analysis of needles on cut-off twigs. Four cut-off twigs were examined. Two of them were supplied with water to protect them against drying (as described above). The other two were left under ambient laboratory conditions with exception that all four twigs were exposed to the regularly alternated dark periods and artificial light periods. Analysis of individually separated needles. Three individual needles were studied in the time-dependent experiments. Two of them were exposed to the dark and light periods as described above for the living trees and cut-off twigs. The last needle was kept in the dark during the two-week experiment to eliminate the effect of artificial lighting. 2.4. Treatment and evaluation of spectra All treatment of spectra was undertaken using the software OPUS NT 4.0 (Bruker). Eight measured spectra were primarily averaged for each of date of
measurement in the case of the time-dependent experiments. The standard deviation curve was also calculated. The averaged spectra and the standard deviation curves were also calculated for needles measured in 10 different points. Every measured and calculated spectrum was cut to the range 3600 – 90 cm21 and exported to JCAMP-DX format. The JCAMP-DX data were imported to The Unscrambler v. 7.8 (Camo, Norway) for the chemometric evaluation. The principal component analysis (PCA) of various data sets was used to obtain the data distribution (‘scores’) along principal components (PCs) and to compare the x-loadings of individual PCs with the measured spectra.
3. Results and discussion 3.1. Analysis of FT-Raman spectra obtained on multiple points of needles The ten FT-Raman spectra obtained for each needle were mutually compared and analyzed using PCA. This comparison was made for each of eight separated needles examined. The sloping baseline shape, especially in the low frequency range (below ca. 1000 cm21), is observed for all the data obtained on the particular needle (for example Fig. 2(A)), while the broad band emission is observed only on one or two points on the needle examined (e.g. Fig. 2(B)). In the case of Raman spectra of needles characterized by a sloping baseline, the scores plots over the first PC (PC1) and the second PC (PC2) as a result of the PCA analysis demonstrate that the sign of the PC1 is related to the side of the needle which the data were collected from (Fig. 3(A)). No outliers were observed in these cases. The xloadings curve of PC1 (Fig. 3(B)) does not show any Raman spectral band, the shape corresponds to the effect of various offsets of the spectra, especially in the range below 3000 cm21. The sloping baseline observed in the spectra below ca. 1000 cm21 is not displayed in the x-loadings of PC1, because the slope is similar in all the spectra recorded. The graph of x-loadings of PC2, which explains only 4% of the variance compared with 93% attributed to PC1, exhibits a contribution of some Raman bands
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Fig. 2. FT-Raman spectra obtained in different places on a needle: (A) ten spectra of a needle I (sloped baseline), (B) ten spectra of a needle II (broad band emission in one place, usual background in the other places). The spectra measured on side 1 of a needle are dotted; the spectra measured on side 2 are full lines.
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Fig. 3. The results of PCA for Raman spectra with sloped baseline measured on one needle in ten different points: (A) scores plot, (B) x-loadings of PC1, (C) x-loadings of PC2. Individual points of scores plot are marked by a code x M, where x ¼ 1; 2 is the side of a needle, M ¼ A, B, C, D, E are the points along the needle on the specified side.
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Fig. 4. The results of PCA for ten Raman spectra of a needle, where a spectrum with broad band emission was measured: (A) scores plot, (B) xloadings of PC1, (C) x-loadings of PC2 Individual points of scores plot are marked by a code x M, where x ¼ 1; 2 is the side of a needle, M ¼ A, B, C, D, E are the points along the needle on the specified side.
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and a weak negative contribution of the sloping baseline below 900 cm21 (Fig. 3(C)). Different scores plots are observed in the case of a needle for which one or two spectra exhibited broad band emission. As shown in Fig. 4(A), such spectra are on the scores plots represented by outlying points. Clustering of the other data is based on the side of the needle, which the spectra were collected from. The shape of the x-loadings of PC1 (Fig. 4(B)) of course corresponds to the difference of the outlier from the other points, i.e. a broad band emission feature is observed. The graph of x-loadings of PC2, which explains only 1% of the variance compared with 99% attributed to PC1, shows a contribution of some Raman bands to the data variability (Fig. 4(C)). This weak effect of Raman spectral bands to the second principal component is the only one evident feature analogous to the PCA results of the data characterized by the sloping baseline. The surface of needles was checked using optical microscopy. The healthy stomatal antechambers
covered by a porous waxy layer can be easily distinguished because the diffuse reflection of white light is observed on these places. No anomalous local effects were observed on the needles, for which the spectra with sloping baseline were collected. On the other hand, abnormal features were observed in the areas (less than 0.8 mm2) of needles, where the spectra with the intense broad emission were recorded. The reflecting waxy layer was damaged, or even it was missing, extraneous black objects were observed on the needle surface, sometimes overlapping the area of the stomatal antechamber (e.g. Fig. 5). In conclusion, the sloping baseline of the FTRaman spectra is an overall feature of some needles. No abnormal effects are observed on the surface of these needles using optical microscopy. The broad emission background is specific for FT-Raman spectra recorded from local areas on the needle surface. The defects on the waxy layer covering stomatal antechambers and/or extraneous impurities are observed in these surface areas.
Fig. 5. Microimage of a needle in the area, which the spectrum with broad band emission was taken from. The stomatal antechambers are marked with circles (diameter of circles ca. 70 mm). The mostly damaged stomatal antechamber is highlighted by an arrow.
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Fig. 6. Averaged FT-Raman spectra of a needle for selected days during the time-dependent experiment performed on an individual needle: (A) day 1, (B) day 3, (C) day 4. (D) day 8, (E) day 14.
3.2. Analysis of FT-Raman spectra of time-dependent experiments Another viewpoint on the spectra with sloping baseline can be based on the results of model
time-dependent experiments performed on individual needles, cut-off twigs and small living trees. A quite significant increase in the slope of the spectral baseline during the two weeks of the experiment was observed for all the separated
Fig. 7. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a cut-off twig kept without external water supply: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
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Fig. 8. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a cut-off twig kept with external water supply: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
needles examined regardless of the lighting schedule (e.g. Fig. 6). The increase of the baseline slope proceeds within the whole duration the experiment. It should be noticed that a decrease of a weak broad band at 3220 cm 21, attributable to a water vibrational mode, can be reliably observed in the first few days.
A slower and weaker increase of the baseline slope was observed in the FT-Raman spectra of needles on the cut-off twigs, which were kept without a water supply (Fig. 7), in comparison with the data from the separated needles. No changes of the spectral background are characteristic for the spectra collected for the needles on the cut-off twigs supplied with water (Fig. 8).
Fig. 9. Averaged spectra of a needle for selected days during the time-dependent experiment performed on a living tree: (A) day 1, (B) day 3, (C) day 4, (D) day 8, (E) day 14.
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At the same time, the intensity of the band at 3220 cm21 is apparently stable during the two-week experiments. Similar effects are observed for the data collected on living trees (Fig. 9). There are no evident changes of the background shape of the spectra during
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the two weeks and also there are no significant changes of the band at 3220 cm21 attributed to water stretching vibrational modes. The trends of the changes in spectral baseline were confirmed on the basis of evaluation of
Fig. 10. Plot of x-loadings for data of a time-dependent experiment on a twig kept without water supply: (A) graph for PC1, (B) graph for PC2. (80 FT-Raman spectra evaluated).
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x-loadings as PCA results, both in the case of experiments on individual separated needles and in the case of twigs kept without a water supply (Fig. 10). The plots of x-loadings of PC1 dominated by the sloped baseline demonstrate the changes of
the baseline slope during the two-week experiments. The variability of Raman bands is then mainly observed on the x-loadings of PC2, but the contribution of PC2 to the overall explained variance is quite minor (5%).
Fig. 11. Plot x-loadings for data of a time-dependent experiment on a twig supplied with water: (A) graph for PC1, (B) graph for PC2. (80 FTRaman spectra evaluated).
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The stability of the baseline noted in the case of twigs with a water supply and in the experiments on living trees was proven on the basis of x-loadings plots for PC1 and PC2. Only Raman spectral bands contribute to x-loadings of PC1 and PC2 for the data collected both on living trees and on twigs supplied with water (Fig. 11). Hence, the water supply eliminates the increase of baseline slope during the time-dependent experiments. Summarizing the data of these time-dependent experiments we can conclude that the increase of the baseline slope is related to the desiccation of the needles. The separated needles become dry faster than the needles on cut-off twigs. The desiccation process is eliminated on living trees or on twigs supplied with water. The moisture of a needle is of course its general characteristics and so its does not significantly depend on its examined part, as was shown in the study of the FT-Raman spectra collected in multiple points on individual needles. The baseline shape is much more sensitive to the moisture of the needle than the intensity of quite weak broad band at 3220 cm21 attributed to water stretching vibrational modes.
4. Conclusions The increase of the background slope was attributed to the effect of drying process of the whole needle. The broad band emission underlying the Raman spectral bands is not a characteristic feature of the whole needles, but it can be ascribed to local defects or impurities on the surface of a needle. The results obtained are important from several points of view. Firstly, it is important to improve the methodology of measurements of standard experiments for environmental monitoring. If the broad band emission is observed, other parts of the needle have to be examined to determine the local source of this effect. On the other hand, if the spectrum with sloping baseline is obtained; there is no reason to perform measurements in multiple points to confirm this shape of the baseline. Secondly, if a spectrum with sloping baseline is recorded, the origin of
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the desiccation has to be found. Hence, the conditions of needle storage have to be checked to eliminate the risk of needle desiccation. Thirdly, the abundance of spectra with either sloping baseline or broad band emission is not uniform for all the forest areas monitored. Hence, in the case of appropriate storage conditions, the shapes of the baseline and the abundance of occurrences of such effects have to be considered in the data evaluation, because these observations are related either to a fraction of dry (dead) needles of the specified age at the locality or to the degree of contamination and/or damage of the surface of needles. Of course, the variation of the baseline shape is not the only effect observed in these experiments. The evaluation of the variability of some Raman bands and its consequences will be the topic of a forthcoming study [7].
Acknowledgements Financial support of the Ministry of Environment of the Czech Republic (grant SI 340/1/01) and of the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 223400008) is gratefully acknowledged.
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