Tree Physiology 24, 1193–1201
© 2004 Heron Publishing—Victoria, Canada
Laser ablation- combustion-GC-IRMS — a new method for online
analysis of intra-annual variation of 13C in tree rings
BIRGIT SCHULZE,1 CHRISTIAN WIRTH,1,2 PETRA LINKE,1 WILLI A. BRAND,1 IRIS
KUHLMANN,1 VIVIANA HORNA1 and ERNST-DETLEF SCHULZE1
1
Max-Planck-Institut für Biogeochemie, P.O. Box 100164, 07701 Jena, Germany
2
Corresponding author ([email protected] or [email protected])
Received November 6, 2003; accepted April 2, 2004; published online September 1, 2004
Summary We present a new, rapid method for high-resolution online determination of δ13C in tree rings, combining laser ablation (LA), combustion (C), gas chromatography (GC)
and isotope ratio mass spectrometry (IRMS) (LA-C-GC-IRMS).
Sample material was extracted every 6 min with a UV-laser
from a tree core, leaving 40-µm-wide holes. Ablated wood dust
was combusted to CO2 at 700 °C, separated from other gases on
a GC column and injected into an isotope ratio mass spectrometer after removal of water vapor. The measurements were
calibrated against an internal and an external standard. The tree
core remained intact and could be used for subsequent dendrochronological and dendrochemical analyses. Cores from two
Scots pine trees (Pinus sylvestris spp. sibirica Lebed.) from
central Siberia were sampled. Inter- and intra-annual patterns
of δ13C in whole-wood and lignin-extracted cores were indistinguishable apart from a constant offset, suggesting that lignin
extraction is unnecessary for our method. Comparison with the
conventional method (microtome slicing, elemental analysis
and IRMS) indicated high accuracy of the LA-C-GC-IRMS
measurements. Patterns of δ13C along three parallel ablation
lines on the same core showed high congruence. A conservative estimate of the precision was ± 0.24‰. Isotopic patterns of
the two Scots pine trees were broadly similar, indicating a signal related to the forest stand’s climate history. The maximum
variation in δ13C over 22 years was about 5‰, ranging from
–27 to –22.3‰. The most obvious pattern was a sharp decline
in δ13C during latewood formation and a rapid increase with
spring early growth. We conclude that the LA-C-GC-IRMS
method will be useful in elucidating short-term climate effects
on the δ13C signal in tree rings.
Keywords: carbon isotopes, cellulose, dendroclimatology, earlywood, fractionation, lignin, palaeoclimatology, Pinus sylvestris, water relations, water-use efficiency.
Introduction
During CO2 fixation, plants discriminate against the heavy
carbon isotope (13C). The magnitude of this discrimination depends on the photosynthetic pathway (C3, C4 or CAM), and on
the ratio of the concentrations of CO2 inside the leaf and in am-
bient air (c i /ca ), which is controlled by stomatal conductance
(g) and assimilation rate (A) (Farquhar et al. 1982, Scheidegger et al. 2000). The A/g ratio is called intrinsic (i.e., plant
regulated) water-use efficiency (WUE) and in C 3 plants, it is
proportional to δ13C of the primary photosynthetic products,
where:
 ( 13 C / 12 C )sample
δ13C = 1000  13 12
 ( C / C )standard

– 1

(1)
Because both A and g are influenced by climate, δ13C of
plant material has been used as a proxy for environmental conditions during assimilation. In particular, the δ13C of cellulose
has been widely used as a proxy of climate record at various
temporal resolutions, because it is progressively deposited in
the annual rings of trees. In addition to climate, post-photosynthetic fractionation associated with downstream primary
and secondary metabolism can alter the isotopic composition
of plant material (Leavitt and Long 1982, 1986, Schleser 1992,
Gleixner et al. 1993, 1998, Jaggi et al. 2002). The signature of
δ13C in tree rings therefore reflects the influences of both climate and intrinsic physiological processes.
Most studies on δ 13C in tree rings have focused on either
long-term trends or interannual variability. Long-term trends
are related to the incorporation of the isotopic signal of CO2
from fossil fuel combustion (de Silva 1979, Freyer and Belacy
1983, Stuiver et al. 1984, Tang et al. 1999) or to shifts in climatic conditions (Zimmermann et al. 1997, Arneth et al. 2002).
Interannual variability of δ13C is related to year-to-year fluctuations in temperature (Wilson and Grinsted 1977), humidity
(Leavitt and Long 1989a, McNulty and Swank 1995), solar radiation (Hanba et al. 1996) and WUE (Dupouey et al. 1993,
Walcroft et al. 1997, Duquesnay et al. 1998, Pate and Arthur
1998). The correlation between climate signals and δ13C of
tree rings seems to be more pronounced at xeric sites than at
mesic sites (Robertson et al. 1997a, 1997b, Kagawa et al. 2003).
Intra-annual sampling of tree rings has usually involved
separating latewood from earlywood to obtain a clear interannual signal (Wilson and Grinsted 1977, Bender and Berge 1982,
Hemming et al. 2001, Jaggi et al. 2002). The isotopic signature
of latewood tends to be closely correlated with climate of the cur-
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SCHULZE ET AL.
rent year, whereas the isotopic signature of earlywood tends to
be closely correlated with climatic conditions of the previous
year (Hill et al. 1995). Furthermore, earlywood of deciduous
species is strongly enriched with 13C despite the relatively high
availability of water during earlywood formation; this has
been related to a selective loss of 12C because of increased
growth respiration during its formation (Schleser et al. 1999b,
Helle and Schleser 2004) or because of the mobilization and
incorporation of 13C-enriched sugar molecules from storage
starch (Schmidt and Gleixner 1998).
High resolution measurements of the variability of δ13C
within a tree ring could provide a link to short-term climatic
events and metabolic processes. A spatial resolution of 200 µm
can be achieved by cutting wood slivers with a razor blade or
scalpel (Wilson and Grinsted 1977, Leavitt and Long 1989b,
Leavitt 1993, Warren et al. 2001, Barbour et al. 2002). Because
temporal resolution increases with ring width at a constant
spatial resolution of the measurement, most studies have been
made with wood from trees with high growth rates. However,
trees growing near their ecological limits are highly sensitive
to changes in climate, but have particularly narrow rings. Analyzing intra-annual variability in such trees requires a high
spatial resolution. A resolution of 40 µm was achieved by drilling minute adjacent holes across the tree ring (Walcroft et al.
1997) or by using a microtome (Ogle and McCormac 1994,
Loader et al. 1995, Helle and Schleser 2004). Unfortunately,
both of these sampling methods are time consuming. Furthermore, the tiny samples are difficult to handle and therefore
prone to cross-contamination during cutting, grinding and
other preparatory steps (Loader et al. 1997).
Here we present a new rapid method for high-resolution,
nondestructive online detection of δ13C from intact biological
samples using UV-laser ablation. We were able to analyze the
carbon isotope pattern in a large number of tree rings with a
spatial resolution of 40 µm. In addition to the speed of analysis, a major advantage of our method is that measurements can
be made on intact cores, thus the core can be used for subsequent dendrochronological measurements or chemical analyses. Our method combines laser ablation (LA) with combustion (C), gas chromatography (GC) and isotope ratio mass
spectrometry (IRMS) (LA-C-GC-IRMS). Unlike other LAGC-MS techniques (Sharp and Cerling 1996, GarbeSchoenberg et al. 1997), ablated wood dust particles are
combusted in a reactor and the resulting gaseous products are
separated on a GC column and then fed into an isotope ratio
mass spectrometer through an open split for online determination of δ13C (Wieser and Brand 1999). In this paper, we focus
on the methodology and discuss the quality of the measurements in terms of precision, reproducibility and comparison
with conventional methods.
spot size and complete ablation pattern were individually controlled and selected using MEO Version 1.4 software (Merchantek-NewWave). The exact location of each ablation spot
was visualized with a camera mounted directly on the laser ablation station. The wood sample was cut into small pieces and,
together with an internal standard, was placed in a cylindrical,
2-ml (15 mm diameter, 10 mm high) sample chamber. The
chamber was covered with quartz glass, which permitted a
good view of the sample and enabled the laser beam to pass
through without significant energy loss. The glass was pressed
onto the chamber by a removable aluminum plate, and an
O-ring between the glass and the chamber ensured an air-tight
seal. The chamber was mounted on a motor-driven, mobile
stage to allow precise positioning of the sample beneath the laser beam. The laser-ablated particles were about 1–2 µm. To
prevent larger dust particles from plugging the capillary system, a 60-µm dust filter (60 µm Netfilter, Millipore, Billerica,
MA) was positioned at the outlet of the sample chamber. The
sample chamber was continuously flushed with helium (He)
(40 ml min –1) and was connected to a micro-combustion furnace by a thin capillary (PEEK, 0.5 mm i.d.). Although a flow
of 40 ml min –1 through the sample chamber was necessary to
ensure adequate transport of the ablation products, the flow
rate had to be reduced before it passed through the combustion
oven. This was achieved by splitting the He flow into a 10 ml
min –1 flow to the combustion oven and a 30 ml min –1 flow to
the atmosphere. For quantitative combustion of the ablation
particles to CO2 and H2O, the sample was passed through an
Al2O3 tube (0.8 mm i.d.) containing CuO wires as a source of
oxygen. The reactor was heated to 700 °C. The CuO had to be
regenerated about every month by flushing oxygen through
the system or by replacing the wires. After combustion, the
gaseous products were passed through a GC column (3.18 mm
diameter and 50 cm long; HayeSep D, Hayes Separations,
Bandera, TX) to separate the sample CO2 from the other gases
and to avoid cross reactions with the CO2 inside the mass spec-
Methods
Figure 1. The laser ablation– combustion line (modified after Wieser
and Brand 1999, John Wiley & Sons, New York. Reproduced with
permission). Wood material is ablated from a whole-wood core with a
UV-laser. Wood dust is combusted over CuO wires at 700 °C. The
gases are then separated on a GC column and water is removed in a
Nafion trap and an additional cold trap. The O2 inlet regenerates the
CuO wires in the combustion furnace. The additional He inlet is used
during changing of the sample.
The laser ablation– combustion interface
The laser ablation–combustion line described by Wieser and
Brand (1999) was modified as shown in Figure 1. For ablation,
we used a frequency quadrupled Nd:YAG 266 nm UV-laser
(Merchantek-NewWave, Fremont, CA). The laser intensity,
TREE PHYSIOLOGY VOLUME 24, 2004
A NEW METHOD FOR δ13C ANALYSIS IN TREE RINGS
trometer ion source. Water originating from the sample and
from the combustion process was removed in a Nafion water
trap (Wieser and Brand 1999). To further increase the efficiency of water removal, we installed an additional water trap
by guiding the capillary through a dewar filled with an ethanol/dry ice slush. Plugging of the capillary was avoided by
taking the capillary out of the dewar and warming it between
runs.
The laser ablation– combustion line was connected to an
isotope ratio mass spectrometer (Delta+ XL, Finnigan MAT,
Bremen, Germany) through an open split interface similar to
the one described by Werner and Brand (2001). The mass
spectrometer could be disconnected from the laser ablation interface by moving the capillary out of the open split. This was
done when a sample was changed and during water removal.
Reference CO2 gas was introduced into the MS by switching a
capillary in and out of the open split tube. In addition, a
low CO2 flow corresponding to a mass 44 signal of 0.5 V
(= 1.5 nA) was added through the open split interface to create
constant conditions in the ion source and reduce drift phenomena during measurement. By saturating active surfaces inside
the ion source of the mass spectrometer, this trickling stream
of CO2 gas flow helped keep conditions constant during measurement.
A rough estimate, based on peak height, IRMS sensitivity
and loss of sample at the open splits, indicated that, of the
0.02 mg of wood material ablated from the UV-laser, 25 – 40%
passed through the system and was available for δ13C measurements.
Laser settings
Spot size, laser intensity and pulse frequency were adjusted to
optimize the trade-off between spatial resolution (spots as
small as possible) and yield of ablated sample material (required to obtain a well-defined peak). For optimal resolution,
we used either a spot size of 40 µm (Figure 2) or ablated lines
of 300 µm in length and 40 µm in width. Ablated lines of these
dimensions yielded an average sample of a specific cell layer,
whereas single spots were affected by local morphological
variations in wood material such as resin ducts. The ablation
process programmed by the laser software and the automated
insertion of the CO2-standard gas triggered by the MS software (Isodat V7.2 and Isodat NT V2.0, Finnigan MAT) were
not coupled and were synchronized manually before each sequence of laser shots.
1195
Figure 2. Photograph of a Pinus sylvestris wood sample showing the
40 µm laser sample shots. The inset represents the spatial arrangement
of laser shots along a tree core. Three lines of holes were divided to
form blocks of eight shots. The movement of the laser (indicated by
the arrows) changes its direction after every block. The individual
holes are slightly displaced in relation to each other forming a zigzag
pattern to increase the spatial resolution.
ently by online combustion of NBS22 (δ13 C = –29.78‰ versus VPDB) on a separate instrument. When the reference CO2
gas values revealed a drift, sample signals were corrected
based on the reference gas information of adjacent peak triplets using the proprietary MS software (Isodat V7.2 and Isodat
NT V2.0, Finnigan MAT). A drift can be caused by changes in
the ionization conditions within the ion source (ion molecule
reactions), by changes in the flow rate or insufficient water removal. An internal standard positioned in the sample chamber
Standards and measurements
An external and an internal standard controlled different processes that potentially cause drift in δ13C values during measurements. The external standard was CO2 gas directly introduced into the mass spectrometer. The internal standard was
placed together with the sample in the sample chamber and
was therefore subjected to the entire process of ablation and
combustion. In between sets of five ablation shots and at the
beginning and end of each run consisting of up to five such
sets, three standard CO2 peaks were introduced as an external
standard into the MS (Figure 3). Standard CO2 gas was obtained from Messer Griesheim, Krefeld, Germany. Its isotopic
composition (δ13 C = –37.89‰) was determined independ-
Figure 3. Measurement scheme. Samples of internal standard (IS) and
CO2 reference gas (Std-CO2) pulses were always measured before and
after measurement of the laser sample shots of wood material (WS).
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SCHULZE ET AL.
revealed possible instabilities in the combustion system, when
loss of oven capacity, plugging or leakage along the sample
path gives rise to fractionation. Three laser shots on the wood
sample were framed by two shots on the internal standard
(Figure 3). Various substances such as glucose, charcoal, marble or synthetics were tested for their suitability as internal
standards but were rejected because they either created heavy
or sticky dust that plugged the ablation– combustion interface,
or were melted by the laser beam and so did not ablate a sufficient amount of dust. Good results were obtained with internode material of a bamboo pole, which seemed to have a constant δ13 C signal over a short distance of 10 mm. The bamboo
was later replaced by C3-cellulose filter paper (IAEA, Austria). During the initial test period, bamboo was an internal
standard, but most of the data presented here were measured
with cellulose filter paper as an internal standard. The bulk values of the internal standards were determined by elemental
analysis (EA) IRMS (Werner and Brand 2001).
Sample material
The sampling site was located in central Siberia (60°43′ N,
89°08′ E). Sample material was obtained from two 95-yearold Siberian Scots pine trees (Pinus sylvestris spp. sibirica
Lebed.) harvested during a field campaign in August 1998
(Trees 16 and 18 of Stand 95 vm (Wirth et al. 2002b, 2002c)).
Tree 18 was a dominant tree with a diameter at breast height
(DBH) of 24.7 cm and a height of 19.5 m. Tree 16 was a
co-dominant tree with a DBH of 19.4 cm and a height of
17.6 m. At the study site, mean annual temperature is –3.6 °C
(1936 –1998), mean monthly air temperature in July and January is 17.3 and –22.9 °C, respectively, and mean annual precipitation is 493 mm, with a growing season precipitation of
300 mm (Wirth et al. 2002a).
Sample preparation
Two adjacent cores (about 150 × 0.5 × 0.5 mm) were cut from
a tree disk (hereafter termed twin cores). One side of each
square core was polished with sandpaper to obtain a smooth
surface with good visibility of individual cells for ablation. A
core thickness of 0.5 mm was sufficient to prevent complete
penetration of the core by the laser beam. To prevent mobile
resins and superficial contaminations during the handling and
cutting process from interfering with the δ13C measurement,
the intact cores were extracted with 2:1 (v/v) toluene:methanol in a Soxhlet extractor (Loader et al. 1997). The organic solvents were removed by drying the core at 70 °C. Solvents did
not affect the experimental results. Samples were stored over
silica under vacuum.
Determinations of the precision and reproducibility of
measurements
Effect of lignin content To test the influence of a varying
lignin content on δ13C, lignin was extracted from an intact core
of Tree 16 (tree rings 1978–1998) following the nondestructive
method of Loader et al. (1997, 2002). The core was kept in sodium chlorite and acetic acid at 40 °C for several days. The extracted, bright-white intact core and the corresponding wholewood twin core were measured by LA-C-GC-IRMS. Because
extraction of lignin led to a shrinking of one of the twin cores, a
pairwise comparison of δ13 C along an absolute spatial scale
was impossible. Instead, the ring width was normalized by calculating the relative position of laser holes within a given ring.
Comparison of LA-C-GC-IRMS with the conventional
EA-IRMS method To compare the δ13C values obtained by
LA-C-GC-IRMS with those obtained by the conventional EAIRMS method, whole-wood material taken from the twin cores
of Tree 18 (tree rings 1978–1998) was measured by LA-CGC-IRMS and with an elemental analyzer (NA 1110, CE instruments, Milan, Italy) connected to an isotope ratio mass spectrometer (Delta +XL, Finnigan MAT). For the EA-IRMS analysis, one of the resin-extracted twin cores was cut with a razor
blade into sections of early- and latewood. If a tree ring was
wide enough, the earlywood was also separated into early
and late sections. Whole-wood material of each section was
weighed in tin cups and measured by EA-IRMS (Werner and
Brand 2001). The whole-wood sections were not ground before EA, because the small sample sizes (0.8 mg) ensured that
samples were combusted quantitatively. The second twin core
was measured by LA-C-GC-IRMS. Because the LA-C-GCIRMS has a higher spatial resolution than the EA-IRMS, several adjacent ablation holes were grouped according to tissue
type and tree ring width to match the spatial resolution of the
EA-IRMS data. The LA-C-GC-IRMS and EA-IRMS data
were compared by Reduced Major Axis regression to account
for variability in both variables.
Measurement reproducibility within the same core To quantify measurement reproducibility, three parallel rows of ablation points or lines were shot radially along the core (tree rings
1941–1961) of Tree 18, yielding three quasi replicate measurements per tissue layer (usually two to five cell rows thick). The
ablation holes forming the rows were shot moving back and
forth in blocks, as shown in the insert to Figure 2, in order to
separate the spatial arrangement of the isotopic composition
from any temporal drift phenomenon.
Inter-tree comparison of measurements We compared the
δ13C patterns of tree rings 1941–1958 of Trees 16 and 18 to
confirm the validity of our measurements. The experimental
design enabled us to distinguish the isotopic signature of morphological features or life history of an individual tree from the
signal related to the climatic conditions of the stand. The time
period was chosen because mean ring width was high in both
trees, permitting a high spatial resolution. Further, the variability of ring widths was high during this period, indicating variable growing conditions that should be mirrored by a high
variability in the δ 13C signal. Because tree ring widths varied
between trees, the position of the laser shots had to be converted to relative instead of absolute positions before the tree
cores could be compared.
Results
Independent measurements of the internal standard material
by EA-IRMS yielded a δ13C of –24.55 ± 0.09‰ (n = 17, errors
are standard deviation throughout) for the cellulose filter paper
and –27.96 ± 0.21‰ (n = 18) for the bamboo internode material. When the cellulose material for the EA-IRMS measurements was reduced to about 0.02 mg, equivalent to the amount
TREE PHYSIOLOGY VOLUME 24, 2004
A NEW METHOD FOR δ13C ANALYSIS IN TREE RINGS
of material ablated by the laser, the standard deviation of the
EA-IRMS values increased to ± 0.31 ‰ (n = 69). Over a
6-month period, the δ13C of the cellulose measured by LA-CGC-IRMS averaged –24.38 ± 0.37‰ (n = 636) (Figure 4) or ±
0.24‰ if the temporal drift was removed by calculating the residual mean square error of a third-order polynomial fit to the
same data. The δ13C of the bamboo averaged –28.28 ± 0.21‰
(n = 202) when determined by LA-C-GC-IRMS. There was a
mean offset of 0.15 ‰ (not significant) for the cellulose and
0.32‰ for the bamboo between the EA-IRMS and LA-C-GCIRMS measurements.
To determine the effect of lignin content on the LA-C-GCIRMS measurements, we compared twin cores (tree rings
1978 –1998) from Tree 16. One core consisted of whole wood
and lignin was extracted from the other core. We observed a
constant offset in δ 13C throughout the 1978 –1998 tree ring series of about 1.5‰, with the extracted core being isotopically
heavier (more positive values of δ 13C) than the intact core
(Figure 5) as a result of removal of the isotopically depleted
lignin. Further, there was strong visible evidence that there
was no difference in the shape of the intra- and interannual
courses of the δ 13C signal. The relative positions of peaks and
troughs in the two cores were generally similar given the introduction of some distortion by the normalization procedure that
was applied to calculate the relative positions of the laser shots
in each tree ring.
Comparing the LA-C-GC-IRMS measurements on wholewood samples of Tree 18 with data obtained by the conventional EA-IRMS method, which involved combustion of resinextracted sections of early-, transition- and latewood in an elemental analyzer, the δ13C series measured by EA-IRMS agreed reasonably well with the mean δ13C series measured by
LA-C-GC-IRMS (Figure 6). The slope of the Reduced Major
Axis, LA-C-GC-IRMS versus EA-IRMS data, was 1.04 with a
95% confidence interval not significantly different from unity
(range of 0.86 to 1.22; Figure 7) However, there was a small
but significant offset of + 0.14‰ (paired t-test, n = 55, P < 0.001),
i.e., the LA-C-GC-IRMS method yielded slightly higher values of δ13C than the EA-IRMS method.
The reproducibility of the measurements within the same
core of Tree 18 was high, with a mean standard deviation
within triplets of ± 0.24‰ (Figure 8). The median of the abso-
Figure 4. Values of δ13C of standard cellulose over a 6-month measurement period. The line denotes the mean value of the standard.
High variation of the values indicates the need for regeneration or replacement of the CuO wires in the furnace oven.
1197
Figure 5. Comparison of δ 13 C of whole wood (solid line) and of
lignin-extracted wood (dashed line) of the twin cores of Tree 16 (tree
rings 1978–1998). Lignin was extracted from intact tree cores by the
nondestructive method of Loader et al. (2002). Because the extraction
leads to shrinkage of the core, the ring width was normalized by calculating the relative positions of the laser holes within the ring to allow comparison of the whole-wood core with the lignin-extracted
core. Vertical lines indicate individual tree rings.
lute range of δ13C within triplets was 0.41‰. There was no
correlation between the standard deviation of δ13C within triplets and its mean (P = 0.94), nor was there a correlation between the standard deviation and distance of the triplet mean
from the average δ13C value of –25‰ from the measured
23 tree rings (P = 0.93). When the laser shots were grouped according to their position within the ring (i.e., earlywood, earlywood–latewood transition, latewood, latewood– earlywood transition), no differences in triplet standard deviations between
the four tissue layers were detected (ANOVA F3; 223 = 0.97, P =
0.44). Over the 23 years, the δ13C values spanned a range of
4.75‰. A minimum δ13C value of –27.02‰ was observed
in the earlywood of Year 1949 and a maximum value of
–22.27‰ occurred in the earlywood–latewood transition of
Year 1957. Within any given ring, the most negative values of
δ13C were measured in the first cell layers of the earlywood,
whereas the least negative values, except in Year 1950, oc-
Figure 6. Comparison of the δ13C series for Tree 18 measured by
LA-C-GC-IRMS method (solid line) and the δ13C series obtained by
the conventional EA-IRMS method (open squares), which involved
combustion of resin-extracted whole-wood sections of earlywood,
transition wood and latewood in an elemental analyzer. Error bars represent the standard deviation within triplets of laser shots. Vertical
gray bars indicate the latewood area of individual tree rings.
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Discussion
Figure 7. Comparison of LA-C-GC-IRMS and EA-IRMS measurements of δ13C in two adjacent cores of Scots pine Tree 18. Values represent δ13 C values of intra-annual layers of tissue in 20 tree rings
(1941–1961). To match the difference in spatial resolution of the two
methods, several laser shots were grouped to represent the intra-annual layers (early-, transition- and latewood), which in the case of the
EA-IRMS method were obtained by manual sample preparation with
a razor blade.
curred at the transition between early- and latewood. The most
rapid changes occurred in the latewood, when δ13C dropped by
up to 3‰ over a distance of 100 µm.
Trees 16 and 18 exhibited a similar interannual pattern of
δ13C with higher values than average in the periods 1943–1945
and 1955–1958 and lower than average values in 1947, 1948
and 1951 (Figure 9). With the exception of Year 1951, the
intra-annual pattern of δ13C was similar irrespective of differences at the interannual scale.
Figure 8. Radial course of δ13C in a core of Scots pine Tree 18 between the years 1941 and 1963. Lines represent three adjacent parallel
traces of laser ablation holes that were shot moving back and forth in
blocks according to the scheme in Figure 2. Vertical gray bars indicate
the latewood area of individual tree rings.
Online laser ablation-combustion-GC-IRMS (LA-C-GC-IRMS)
is a fast and reliable method for measuring δ 13C in solid biological samples. Our current measurement scheme allows us
to take a sample (i.e., a laser shot) every 6 to 8 min. This procedure leaves the cores intact (Figure 2), providing the opportunity to study and relate the anatomical features of the wood
(cell type, cell size, width of cell wall, etc.) in the vicinity of individual ablation holes to isotopic composition. The core or
sections of the core can also be used for dendrochronological
investigations, X-ray densitometry, radiocarbon dating and
dendrochemical analyses. At the same time, the LA-C-GCIRMS method avoids several disadvantages of the conventional EA-IRMS method which uses thin wood sections, including time-consuming sample preparation and the danger of
cross-contamination during the tedious and difficult handling
of tiny samples from microtome sectioning, homogenization
and extraction (Loader et al. 2002). Furthermore, the diameter
of typical thin slivers are at least as large as the core diameter.
If the orientation of the cell rows is not perpendicular to the
core, the microtome cuts through several cell layers, smearing
the temporal/spatial signal. With the LA-C-GC-IRMS method, sample size can be controlled by adjusting the laser frequency and intensity, and it is also possible to adjust the laser
path to the curvature of the cell rows.
The twofold lower variability in δ13C of the cellulose filter
paper compared with the bamboo internode illustrates the superiority of cellulose filter paper as an internal standard. Standard deviation of routine EA-IRMS measurements of standard
material was about three times lower than measurements with
LA-C-GC-IRMS. However, when the amount of the sample
material was reduced to an amount equivalent to the yield of
the laser ablation, the standard deviation of the EA-IRMS measurements was slightly higher than the standard deviation of
the LA-C-GC-IRMS measurements. Thus, the LA-C-GC-IRMS
technique shows good precision for detection of intra-annual
variability in tree rings. The drift in the internal standard values during a measuring period of about 6 months revealed an
unidentified fractionation step of the sample gas during pas-
Figure 9. Comparison of the radial course of δ13C in cores from two
trees from the same stand for the 18-year period between 1941 and
1958. Tree 18 (solid line) was a dominant tree and Tree 16 (dashed line)
was a codominant tree. Vertical lines indicate individual tree rings.
TREE PHYSIOLOGY VOLUME 24, 2004
A NEW METHOD FOR δ13C ANALYSIS IN TREE RINGS
sage through the system (Figure 4). We have been unable to
clarify the nature of this additional discrimination step. It may
be related to plugging or a leak in the system, although loss of
reactor oxidation capacity or insufficient water removal could
also contribute to such fractionation. However, the exact magnitude of the bias has been quantified and corrected for. A
gradual increase in the variation of δ13C of the internal standard indicated the need to regenerate the reactor or replace and
oxidize the Cu wires. In general, the measurements of the cellulose filter paper showed high stability of the system over a
measuring period of about 2.5 h.
To exploit the full potential of the LA-C-GC-IRMS method,
in terms of increased sample throughput, and to allow subsequent investigations of the core, whole-wood samples should
be used. Previously, the majority of dendroclimatological isotope studies have extracted cellulose from tree rings before
analysis for several reasons. Whole wood is a complex mixture
of substances that differ in their δ 13C values. Lignin, which is a
product of secondary metabolism is, on average, depleted by
2 to 4‰ relative to cellulose (Benner et al. 1987). Therefore,
any substantial change in the ratio of cellulose and lignin inevitably leads to a shift in δ 13C, obscuring the isotopic signal of
discrimination during C fixation. Furthermore, it has been reported that lignin is deposited in cell walls toward the end of
the growing season (Kozlowski and Pallardy 1997), suggesting that it is built from photosynthates that were formed later
than those used to produce the cellulose of the same cell.
Bulk wood analysis makes comparison of samples from other
stands difficult, because stand conditions influence wood composition. Nonetheless, several studies have shown that, for the
same material, the isotopic offset between cellulose and wholewood measurements is constant (Wilson and Grinsted 1977,
Livingston and Spittlehouse 1996, Leuenberger et al. 1998,
Schleser et al. 1999a, Loader et al. 2003). In addition, analyses
of the ultrastructure and chemical composition of wood suggest that the differences in the lignin to cellulose ratio between
early- and latewood are too small to cause a significant shift in
the isotopic signal (Walcroft et al. 1997). Based on data on volume share and lignin concentration of three cell wall compartments (secondary cell wall, middle lamella and cell corner) in
both early- and latewood (Sjöström 1993, Gindl 2001), a theoretical seasonal decline of 0.08‰ can be attributed to ultrastructural differences. This effect is small compared with the
magnitude of the seasonal oscillation that covers a range of almost 5‰. Given these findings and the near-constant offset between whole-wood and cellulose samples in our study, we recommend that only mobile substances be removed from the
cores. If cellulose must be extracted from the cores, we recommend the cellulose extraction method described by Loader et
al. (1997, 2002). These authors present a method for extracting
cellulose from more or less intact cores that can be probed by
laser ablation. However, because even this careful extraction
method distorts the cellular dimensions, the possibility of performing subsequent anatomical analyses is limited.
Compared with the EA-IRMS method, the accuracy of the
LA-C-GC-IRMS method was high, notwithstanding that matching of the two data sets for statistical comparison was complicated by the different spatial resolutions, such that several
laser shots had to be averaged to represent the thick tree ring
1199
subsections measured by EA-IRMS. The LA technique facilitated analysis of minute spatial differences within the wood
material, which maybe responsible for some of the scatter in
Figure 7. The EA-IRMS method averages this important information. Comparison of δ13 C values in adjacent lines of ablation shots in Tree 18 revealed a remarkably parallel course of
the three lines (Figure 8). However, the neighboring three
shots do not represent true replicates for two reasons. First,
tangential variability cannot be excluded, even at this small
spatial scale. Second, the cell rows are not always parallel, or
of constant width. It is therefore not always possible to position a triplet exactly within a homogeneous tissue layer.
The same congruence between isotopic patterns was observed when comparing two whole wood cores taken from two
trees (Tree 16 and 18) in the same stand (Figure 9). The range
of values within the smaller codominant Tree 16 was slightly
larger than those in the dominant Tree 18. The similarity of the
intra- and interannual courses of the δ 13C values between trees
confirmed that the observed signal is predominately influenced by stand growth conditions, not individual features of
the morphology and physiology of the two trees.
The intra-annual pattern of δ 13C followed the typical course
for coniferous species with a gradual increase in δ 13C in the
earlywood and a sharp decline during latewood formation.
Similar intra-annual patterns of δ13C values in tree rings have
been reported for other coniferous species: Pinus radiata
D. Don (Walcroft et al. 1997, Warren et al. 2001, Barbour et al.
2002), Pinus strobus L. (Leavitt 1993), Pinus pinaster Ait.
(Warren et al. 2001), Pinus ponderosa (Leavitt and Long
1991), Abies kawakamii (Hayata) Ito (Sheu et al. 1996) and
Picea abies (L.) Karst. (unpublished observations, G.H.
Schleser, Forschungszentrum Jülich, Germany). There is general agreement that this δ13C signature reflects the interaction
between micrometeorological variables and soil water availability (Barbour et al. 2002). In contrast, many deciduous species exhibit a decrease in δ13C (more positive values) in
earlywood that is unrelated to climate (Populus nigra L.,
Fagus sylvatica L., Quercus petraea (Matt.) Liebl., Morus
alba L.; Schleser et al. (1999b), Helle and Schleser (2004)).
Carryover effects from photosynthates fixed in the previous
growing season and discrimination along with high rates of
growth respiration during rapid earlywood formation have
been proposed as possible mechanisms (Schleser 1992,
Schleser et al. 1999b, Damesin and Lelarge 2003, Helle and
Schleser 2004). Ring-porous broadleaf trees rely on the rapid
formation of large earlywood vessels, because most old vessels are cavitated by the end of the winter (Kozlowski and
Pallardy 1997). In contrast, in conifers, the earlywood is usually assimilated directly from the currently fixed carbon and
the gradual start of earlywood formation does not require high
physiological activity (Dickmann and Kozlowsk 1970, Glerum 1980).
The major application of LA-C-GC-IRMS is in high-resolution dendroclimatology and plant physiology. In wide tree
rings, individual laser ablation holes can represent tissue layers formed within a few days. Barbour et al. (2002) were able
to relate short-term climatic events such as drought or high
rainfall to the isotopic signature of tissue layers formed within
3 days to 3 weeks. However, transforming the spatial sequence
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1200
SCHULZE ET AL.
of laser holes into a temporal scale is not trivial. Although
some older studies relied on visual comparison of the relative
position of peaks and troughs in isotope and climate data
(Loader et al. 1995), two more recent studies used material
from trees with known or modeled growth rates (Walcroft et al.
1997, Barbour et al. 2002). Exact growth rates can be known
only if the diameter increment has been recorded with dendrometer bands. Because such direct growth measurements are
scarce, it is necessary to use proxies for growth rates. The most
promising proxies are anatomical parameters like cell number,
cell dimensions, cell wall thickness and tissue density. Vysotskaya et al. (1985) presented a procedure of converting
cell-size measurements to growth rate estimates. Once calibrated for a certain site, such transfer functions can provide a
time-match between δ13C and real time climate data based on
cell size arrays of the same core.
At this early stage of method development, we focused primarily on precision rather than speed, so there remains potential for large improvement. Currently, the rate-limiting step of
the measurement process is the separation of gases in the GC
column. We are analyzing the performance of GC columns
that can operate at higher carrier gas flow rates. Furthermore,
the sample chamber design can be improved to fit the elongated shape of tree cores. This would minimize the air volume
around the sample core, reducing the time required to flush the
chamber and allow for more sample shots in a sequence. Recent publications have emphasized the importance of simultaneous measurements of δ 13 C and δ 18 O, because the relative
responses of these two ratios can be related to plant sensitivity
to evaporative conditions (Barbour et al. 2000, 2002, Saurer
2003). Measurement of δ18 O by LA-C-GC-IRMS is possible
in principle, but will require substantial modifications of the
interface, including installation of a high-temperature pyrolysis reactor with a carbon-dominated oxygen-free environment,
which is a technical challenge. Nonetheless, our initial data
show a high potential of the laser ablation methodology that
could form the basis of a link between tree ring chronology
and climate research at the cellular level.
Acknowledgments
We gratefully acknowledge the support from Finnigan MAT in Bremen who generously provided the prototype interface for this study,
originally developed by Wieser and Brand. We thank Heike Geilmann
for measuring the EA-IRMS samples and Roland Werner from the
Isolab at the Max Planck Institute for Biogeochemistry for many
hours of help, troubleshooting and fruitful discussions.
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