JXB Advance Access published online on February 24, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm007
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles
í Janá
ek3
1Charles University in Prague, Faculty of Science, Department of Plant Physiology, Vininá 5, CZ-12844, Prague 2, Czech Republic
2Institute of Botany, Academy of Sciences of the Czech Republic, CZ-25246 Pr
honice, Czech Republic
3Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Víde
ská 1083, CZ-14220, Prague 4, Czech Republic
* To whom correspondence should be addressed. E-mail: albrecht{at}natur.cuni.cz
Received 22 June 2006; Revised 18 December 2006 Accepted 22 December 2006
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Abstract |
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Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.
Key words: Confocal microscopy, disector method, fakir method, internal surface area, mesophyll, Norway spruce, stereology
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Internal leaf structure, which is connected with the process of photosynthesis because it influences the interception of light and diffusion of CO2, is known to be affected by the environment. Recent studies show that relationships between leaf anatomy parameters and photosynthesis are important in leaf acclimation to high or low irradiances (Pandey and Kushwaha, 2005) or elevated CO2 concentrations (Eguchi et al., 2004).
The first quantitative descriptions of leaf anatomy (Pazourek, 1988) were based on counting planar features, especially counting stomata per unit area of the leaf (stomata density). In the first half of the 20th century, methods for quantifying internal leaf structure, such as cell volume and cell surface area per unit volume of leaf tissue, emerged (Turrell, 1936). These methods were model based, i.e. they assumed that mesophyll cells have specific shapes of simple geometric bodies. However, such model-based methods are usually biased because the shape of mesophyll cells is often irregular, thus strongly deviating from the model geometrical bodies, and so the measurements can lead to unreliable and imprecise results.
Development of stereological methods during the second half of the 20th century brought new approaches that could be applied to quantitative analysis of internal leaf structure (Weibel, 1979; Howard and Reed, 1998). The stereological point-counting method for measuring volume density or volumetric proportion of leaf tissues has become widely used (Chabot and Chabot, 1977; Pazourek, 1977; Parkhurst, 1982; Albrechtová and Kubínová, 1991; Kubínová, 1991, 1993).
Corresponding stereological methods were established for surface area measurements, such as cell surface area exposed to intercellular spaces [usually expressed per unit leaf area, A(mes)/A]; however, some assumptions about the cell shape were often still made, for example, a specific shape factor for the given population of cells had to be applied (Parkhurst, 1982; Slaton and Smith, 2002; Rhizoupoulou and Psaras, 2003).
Design-based, assumption-free stereological methods, enabling unbiased evaluation of the structure of three-dimensional (3D) objects of arbitrary shapes, however, were developed and adapted to evaluate leaf tissues. The proposal to estimate the mesophyll cell surface area exposed to intercellular spaces (i.e. internal leaf surface area) using a design-based stereological method of vertical sections (Kubínová, 1991, 1993) was not used by other authors.
The situation is similar for counting mesophyll cells or measuring their mean volume. Most developed methods were again model based (Chonan, 1965; Wilson and Cooper, 1967; Sasahara, 1982), or the number of cells was determined by counting isolated cells (Maksymowych, 1959; Sasahara, 1982; Lieckfeldt, 1989), which may lead to the loss of cells during the manipulation of the cell suspension or by damage to certain cells. The design-based stereological disector method (Sterio, 1984) has not found wide application in plant anatomical studies, although its application to count mesophyll cells has been worked out (Albrechtová and Kubínová, 1991; Kubínová, 1991, 1993).
Considering that the reliable but laborious methods proposed in the 1990s have only rarely been applied, there is a clear need for less demanding, efficient, and unbiased methods for measuring physiologically important mesophyll anatomical characteristics, such as internal leaf surface area, mean mesophyll cell volume, and cell number in the leaf. The recently developed methods, based on confocal stereology, appear to be suitable for this purpose because they are efficient, unbiased, and can be applied to thick fresh tissue sections, thus minimizing time spent on tissue specimen preparation.
Confocal stereology is a contemporary approach that evaluates structures using a combination of stereological methods and confocal microscopy (Pawley, 1995), enabling perfectly registered stacks of thin serial optical sections (
350 nm thick) within thick specimens to be obtained. Digital images of such stacks represent suitable 3D image data for quantitative measurements. Howard et al. (1985) presented the first application of confocal microscopy to stereological measurements in their concept of an unbiased sampling brick. Confocal microscopy proved to be useful especially in the application of stereological methods based on spatial estimators that evaluate small 3D samples of the structure under study (Howard et al., 1985; Howard and Sandau, 1992; Kubínová and Janáek, 1998; Kubínová et al., 1999, 2002). A 3D sample of examined tissue can be analysed if a rectangle within a microscope's field of view is focused through. Using special software, different virtual test probes with an arbitrary predefined (e.g. random) position and orientation can be generated within the stack of sections and can be applied directly to these 3D image data. This study presents the confocal stereological methods used to evaluate the mesophyll structure of narrow leaves, such as conifer needles. The mean mesophyll cell volume and cell number in a needle are estimated using the optical disector method (Gundersen, 1986), and the internal needle surface area (defined as the surface area of mesophyll cell walls exposed to intercellular spaces) using the fakir method (Kubínová and Janáek, 1998). Unlike classical stereological methods applied to thin physical sections, the fakir method does not require randomization of the orientation of the section, hence the physical thick sections can be cut in an arbitrary direction. Therefore, the slices were cut perpendicular to the main axis of the needle, which is most suitable from the technical point of view.
Further, it was checked whether classical stereological methods can also be applied to images captured using confocal microscopy. The implementation of the point-counting method (Weibel, 1979) and Cavalieri principle (Gundersen and Jensen, 1987) for estimating the needle volume, volume density of needle tissues, and their cross-sectional areas is presented.
The aims of the present study include the following: (i) to test the usability of thick fresh needle sections cut using a hand microtome without any pre-processing for confocal microscopy and subsequent application of stereological methods applied to a series of optical sections; (ii) to estimate the stereological parameters that characterize the anatomical structure of mesophyll of Norway spruce needles; and (iii) to find out if the selected anatomical parameters are able to capture subtle changes in needle anatomy induced by acid rain treatment.
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Materials and methods |
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Plant material and simulated acid rain treatment
Sun-exposed needles from the upper whorl of one potted 5-year-old Norway spruce (Picea abies L. Karst.) were used as plant material for this study. The tree was located in the botanical garden of the Department of Plant Physiology, Charles University in Prague. The first batch of five first-year needles from one branch of the experimental tree was sampled in January 1999. These needles were used as an untreated control sample. After needle sampling, the tree was treated for 1 year with a solution (pH 2.9 containing 66.0 mg dm–3 SO
, 18.6 mg dm–3 NO
, 6.0 mg dm–3 NH
) to simulate the precipitation quality of acid rain (SAR) monitored in the Krkono
e Mountains at the beginning of the 1990s (Vávra, 1992). The second batch of five first-year needles from the same branch was sampled in January 2000 after 1 year of treatment.
Sample preparation and confocal microscopy
Thick transverse needle sections were cut in positions sampled according to the principle of systematic uniform random sampling (Kubínová, 1991), as shown in Fig. 1A, immediately after needle collection. Because needle length, which was measured first, was 10 mm in both variants, the interval between sampled sections was chosen to be 2 mm, resulting in five thick sections per needle available for further analysis. The sections, 60 µm thick, were cut using a hand microtome. A position, z, of the first section was randomly selected within the 2 mm interval (Fig. 1A). Sections were placed into distilled water between two cover glasses without any staining or other pre-processing—only autofluorescence of chlorophylls and phenolic compounds in the cell walls was exploited.
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A series of 40 optical sections, 1 µm apart (Fig. 1B), was acquired using a Bio-Rad MRC600 laser scanning confocal microscope using excitation with the Kr/Ar laser at a wavelength of 488 nm and an emission range above 515 nm, and a Nikon water immersion planapochromat objective (x60, NA=1.2) to ensure that the refractive indices of the tissue and the immersion fluid matched. On average, 22 fields of view (211 µmx141 µm), selected using the systematic uniform random sampling (Fig. 1C, see also Fig. 7 in Kubínová, 1994), were captured per needle. The positions of these fields of view were set using Modig 105 potentiometric sensors (Megatron Elektronik AG & Co., Munich, Germany) attached to the microscope stage.
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To measure needle volume and the volume density of mesophyll (i.e. mesophyll volume per unit volume of the needle) and other needle tissues, images of entire needle sections (Fig. 2) were collected using a x4 Nikon dry planachromat objective (NA=0.1).
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Stereological methods for measuring needle structural parameters
The images of whole systematic sections of the needle (Fig. 2) were used to measure needle volume according to the Cavalieri principle (Gundersen and Jensen, 1987; Kubínová, 1993), where the total area of needle sections was multiplied by the distance between neighbouring sections. The images were also used to measure the volume density of mesophyll according to the point-counting method (Weibel, 1979; Kubínová, 1993). At the same time, the point-counting method was applied to the estimation of cross-sectional areas of the needle, mesophyll, central cylinder, and epidermis, together with hypodermis at different positions along the needle axis.
For all remaining measurements, the stacks of serial confocal sections, sampled as shown in Fig. 1C, were used to provide a higher lateral and axial resolution needed to evaluate the more subtle mesophyll structures.
First, the optical disector method was applied, yielding the number of mesophyll cells in a needle, mean mesophyll cell volume, and the ratio of intercellular spaces per mesophyll cell volume. The FAKIR and SANDAU programs written in TurboPascal 6 for IBM PC under the MS DOS operating system (available as a freeware via Internet at website http://www.biomed.cas.cz/fgu/fakir/3dtools.htm) or the DISECTOR module running in the Ellipse (ViDiTo, Slovakia) environment were used.
The optical disector (Gundersen, 1986) is based on focusing through the 3D probe within a thick section. Think of it as a box-shaped piece cut from jelly filled with raisins; some of the raisins are cut by the cover, sides, and bottom of the box. The whole raisins that are lying within the box are certainly counted but what about those that were cut in two by the box sides? The proper method is to count only those that are intersected by the bottom, right, and back of the box, and exclude those intersected by the remaining three sides. In the present case, where there is a thick needle section with mesophyll cells instead of jelly with raisins, a counting frame can be placed on the needle slice and, by focusing through the tissue, the ‘virtual’ box of tissue is obtained. The contents of the box of tissue can then be viewed by browsing through a stack of optical sections captured by a confocal microscope. During focusing up from the bottom, only those mesophyll cells within the box that disappear during focusing (i.e. those not intersected by the upper side) and at the same time are not intersected by the left and front sides of the virtual box (i.e. their sections are not intersected by the exclusion lines of the unbiased counting frame, see Fig. 3) are counted. After applying the disector probes in all sampled stacks of confocal sections in each needle (Fig. 1C), the total number of mesophyll cells in the needle, N(mes.cell), was estimated using the formula:
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The mean mesophyll cell volume in the needle was estimated using a similar measurement with the same disector probes:
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The ratio of intercellular spaces per mesophyll cell volume was measured using a point-counting method utilizing the same point grid and test frames selected for the disector measurements applied to one level of each disector box. The test points falling within the intercellular spaces were counted and divided by the number of test points found in the mesophyll cells.
The internal surface area of the needle, S(int), was estimated using the fakir method (Kubínová and Janáek, 1998). The fakir method uses systematic probes consisting of parallel test lines (resembling nails of a fakir bed piercing the surface). When estimating the internal surface area of the needle, S(int), the intersections between the mesophyll cell walls exposed to intercellular spaces and the fakir probes were counted (Fig. 4). It can be imagined that the cells were pierced through by the nails of the fakir bed and the number of times the nails went into or out of the exposed cell walls was counted. To increase the efficiency of the measurement, a cubic spatial grid consisting of three mutually perpendicular fakir probes was used, halfway shifted with respect to each other (Fig. 5; Kubínová and Janáek, 1998). The internal surface area of the needle, S(int), was then estimated using the formula:
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The above parameters of needles before and after 1 year of acid rain treatment were compared. Further, selected parameters were compared with respect to the positions of transverse sections along the needle longitudinal axis to see if they are constant or variable along the needle.
Statistical analysis was performed using a paired t-test and one-way ANOVA, 
level=0.05, and a Tukey–Kramer multiple comparison test if the data were normally distributed; otherwise the Kruskal–Wallis Z-test was applied.
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Results |
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According to the tests with needle transverse sections of different thickness, 60 µm thick sections were found to be optimal for capturing a series of 40 optical sections 1 µm apart with sufficiently high resolution to conduct stereological measurements (Fig. 1B). Thinner sections would not enable the minimalization of the effect of possible distortions of section-cutting surfaces. Sections thicker than 80 µm, however, caused problems with image acquisition when using the confocal microscope due to the limited working distance of the water immersion objective used.
Results for stereological parameters of the Norway spruce needles and their mesophyll anatomical structure obtained using this study's novel stereological methods are shown in Table 1. The comparison of selected mesophyll parameters was conducted to evaluate the effect of simulated acid rain on current Norway spruce needles. Although the needle length did not differ between treated and untreated needles, needle volume significantly decreased after SAR application due to the decrease of cross-sectional area of treated needles (Fig. 2). The volume density of mesophyll did not change significantly after the treatment, while the volume of intercellular spaces per mesophyll cell volume increased, which corresponded well to the decrease in the number of mesophyll cells per unit volume of the needle. Also the total number of mesophyll cells decreased in treated needles. No significant change in mean mesophyll cell volume was observed. The internal surface area of a needle decreased after the SAR application, but the internal needle surface area per unit needle volume increased (Table 1).
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Further, selected needle anatomical parameters obtained using classical stereological methods in cross-sections cut at different positions along the needle longitudinal axis were compared (Fig. 6). Needle and mesophyll cross-sectional area were significantly higher in the middle positions of the needle than at the base and apex, while the area of epidermis and hypodermis or central cylinder did not display significant differences with respect to the cross-section position. The number of mesophyll cells per unit needle volume and mean mesophyll cell volume did not differ between the cross-section positions (Fig. 7). The effect of the cross-section position on the ratio of intercellular spaces per mesophyll cell volume and internal surface density was also tested, but the data showed very high variability in the sample studied and thus the results were not found to be reliable.
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Discussion |
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The present study proved the feasibility of methods of confocal stereology for efficient measurement of stereological parameters characterizing the anatomical structure of Norway spruce needles. It was shown that not only can the recently developed methods based on evaluation of 3D image data be successfully applied to needle structure analysis but also classical stereological methods can be easily applied to images captured using confocal microscopy. The present methods can be directly used for quantitative anatomical studies of other coniferous needles and other types of narrow leaves and, after slight modifications, also for the analysis of leaves of different shapes, for example, broad bifacial leaves where different sampling of leaf segments and transverse thick sections would be applied, as indicated in Kubínová (1993). The methods can also be easily modified to evaluate other plant organs, such as roots and stems.
Thin optical sections can be acquired in conventional light microscopy when using a lens with a high numerical aperture; however, observation of deeper layers in fresh thick sections is not possible due to the blur from out-of-focus specimen layers. If an objective with a low numerical aperture, i.e. a higher depth of focus, is used, several cell layers are displayed on the final observed projection, which also makes measurements difficult. Confocal microscopy enables the acquisition of thin optical sections within a thick physical section; thus, details of tissue arrangement are revealed. Plant material, containing autofluorescent components, is very suitable for fluorescence microscopy in plant anatomy studies even without any special staining. In leaves, the autofluorescence of chloroplasts containing photosynthetic pigments and of phenolic compounds localized either in cell walls or in vacuoles abundant in certain leaves, such as needles of Norway spruce (Soukupová et al., 2000), can be exploited.
Most studies on foliar internal structure use sections of fixed and embedded plant material (Turrell, 1936; Eguchi et al., 2004; Pandey and Kushwaha, 2005); however, during fixation or embedding, tissue deformation and other artefacts may occur (Uwins et al., 1993). Therefore, another advantage of using free-hand sections of fresh needles is the elimination of such deformation problems. Distortions of cutting surfaces of transversely cut needle sections, however, are likely to occur because conifer needles exhibit the arrangement of mesophyll cells in transversally oriented interconnected layers (Esau, 1953). The location of the disector and fakir probes within the stack of optical sections could play a role in overcoming the observed variability, and will be tested further. Using fresh material can limit the application of the method in experimental research because only a few needles can be analysed simultaneously—processing of one needle including sectioning and image acquisition took 4 h in the present study. This problem could be solved if a suitable form of storage of sampled material is found. The easiest way would be freezing, provided the thawing does not cause structural deformations, which is now being studied, as is the effect of possible deformations due to the cutting of unembedded material.
In many physiological studies conducted on conifers, only cross-sections from the middle part of the needle are used to measure needle and mesophyll geometrical parameters (e.g. Apple et al., 2002), without checking that this specific position is representative for the entire needle. The differences in the cross-sectional area along the needle axis were shown in a recent study (Lhotáková et al., 2006), when parameters of cross-sections sampled in the middle part of the needle differed from those sampled at the tip and base. In the present study, however, special mesophyll structural parameters, such as the number of mesophyll cells per needle volume and mean mesophyll cell volume, did not change with the cross-section position. Zwieniecki et al. (2006) also observed constant needle parameters along 80% of the needle axis on needles of three pine species. Thus, the evaluation of sections taken only in the middle part of the needle can be sufficient to obtain reliable results, which is useful especially in more extensive physiological studies. However, the variability along the needle axis should be verified for each specific anatomical parameter and variant.
A model-based approach, when assumptions about mesophyll cell shapes are made, is still commonly used to determine mesophyll structural parameters (for recent applications, see Slaton and Smith, 2002; Rhizoupoulou and Psaras, 2003; Barbour and Farquhar, 2003; Pandey and Kushwaha, 2005). Some of the pitfalls of approximating cells by geometrical solids were pointed out by Kubínová (1993, 1994) and Slaton and Smith (2002), stressing inaccuracy especially in surface area measurements. Norway spruce mesophyll cells have an irregular lobed shape, and thus modelling them with cylinders or other geometrical bodies is not appropriate, and yields biased results. Therefore, only design-based methods appear to be suitable for unbiased estimation of mesophyll geometrical parameters. Many of the difficulties connected with the implementation of classical design-based stereological methods for estimating needle mesophyll parameters are overcome by the presented approach using confocal microscopy, also resulting in much faster measurements. In the case of internal needle surface area measurement, the analysis was more than three times faster when compared with the implementation of the method of vertical sections (Kubínová, 1991, 1993). The time efficiency of the new approach is further increased by the possibility of using the same stacks of confocal sections for measurements of different parameters, i.e. mean mesophyll cell volume, mesophyll cell number in a needle, and internal surface area of needles, as well as the ratio of intercellular spaces per mesophyll cell volume.
Only a few studies using stereological methods based on 3D probes, such as a disector, to obtain a detailed description of internal leaf structure have been published. The optical disector method was successfully applied to paraffin sections of grass leaves when a 3D disector probe was placed by optical sectioning within one 20 µm thick physical section (Albrechtová and Kubínová, 1991; Kubínová, 1991). To our knowledge, the fakir method has yet to be used in any plant anatomy study.
Conifer needle anatomy has been predominantly studied for Norway spruce since the 1980s, mainly due to the widespread forest decline caused by atmospheric pollution and acid rain (Albrechtová et al., 2001). However, most studies describe the anatomical alterations only qualitatively, for example, by the presence of collapsed cells (Kukkola et al., 2005) or loss of chloroplasts (Moss et al., 1998). In the present study, the purpose was to test if the confocal stereology analysis is capable of capturing subtle changes in mesophyll arrangement. To reduce variability of plant material, it was decided to analyse needles only from the same branch. To induce the subtle changes, a treatment with simulated acid rain was used as a stress factor, which is known to cause damage in the mesophyll with already described symptoms, such as a decrease in needle length and volume or an increase of the volume of the intercellular spaces (Masuch et al., 1992). The significant decrease of needle volume was detected as the effect of SAR treatment, which was caused by a reduced area of the needle cross-sections, while needle length remained unchanged. Needle thinning, expressed as lower cross-sectional area, was already documented in Norway spruce needles collected from a heavily polluted site in comparison with needles collected from a healthy site (Masuch et al., 1992). Similarly, as reported by Masuch et al. (1992), the volume of intercellular spaces per mesophyll cell volume increased after acid rain application. By contrast, Kukkola et al. (2005), who assessed the proportion of intercellular spaces using the point-counting method on semi-thin sections of Scots pine needles treated with simulated acid rain, found no effect of SAR on the extent of intercellular spaces. Moss et al. (1998), however, detected large cavities in red spruce mesophyll caused by the acid mist event, which corresponds to the present finding of increased volume of intercellular spaces after the SAR treatment.
Differences in mesophyll architecture, such as leaf porosity expressed as volume percentage of intercellular spaces in mesophyll, are important for evaluating the impact of internal leaf surface area or chloroplast area available for CO2 absorption during photosynthesis (Slaton and Smith, 2002). The relationships between the structural arrangement of mesophyll and leaf reflectance could bring new tools for monitoring photosynthetic performance using remote sensing methods. Slaton et al. (2001) found a strong correlation between the ratio of mesophyll cell surface area exposed to intercellular air spaces expressed per unit leaf area and reflectance in near infrared (800 nm) for 48 alpine herbaceous species.
Models of major features of leaf photosynthesis based on mathematical description of leaf gas exchange, electron transport, and biochemistry that were developed in the last three decades stimulated development in understanding the physiology of photosynthesis and methodology of photosynthetic measurements (Farquhar et al., 2001). The modelling of canopy photosynthesis, however, also requires more detailed knowledge on relationships between photon interactions and leaf structure, leaf orientation, and photosynthetic potential, which can be obtained from a quantitative description of a 3D structure of plant tissue by using simulation (Ustin et al., 2001). This recent development in interpretation of structure–function relationships in a leaf indicates that quantitative ultrastructural studies, similar to those presented herein, are of fundamental importance in understanding photosynthesis at the leaf and canopy level. Thus, detailed and unbiased estimators of mesophyll parameters are becoming crucial for modelling physiological processes that underlie photosynthesis, such as gas transport (Aalto and Juurola, 2002; Juurola et al., 2005) or radiative transfer (Ustin et al., 2001). It is believed that the present stereological analysis is a promising new tool for plant physiological and modelling research.
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Acknowledgements |
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Supported by the Ministry of Youth and Education of the Czech Republic (grant LC06063), and the Academy of Sciences of the Czech Republic (grants A5011810, A600110507, and AV0Z50110509). We thank Janice Forry for proofreading the manuscript and help with the English.
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