Most cited article - PubMed ID 17322549
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
Imaging spectroscopy of vegetation requires methods for scaling and generalizing optical signals that are reflected, transmitted and emitted in the solar wavelength domain from single leaves and observed at the level of canopies by proximal sensing, airborne and satellite spectroradiometers. The upscaling embedded in imaging spectroscopy retrievals and validations of plant biochemical and structural traits is challenged by natural variability and measurement uncertainties. Sources of the leaf-to-canopy upscaling variability and uncertainties are reviewed with respect to: (1) implementation of retrieval algorithms and (2) their parameterization and validation of quantitative products through in situ field measurements. The challenges are outlined and discussed for empirical and physical leaf and canopy radiative transfer modelling components, considering both forward and inverse modes. Discussion on optical remote sensing validation schemes includes also description of a multiscale validation concept and its advantages. Impacts of intraspecific and interspecific variability on collected field and laboratory measurements of leaf biochemical traits and optical properties are demonstrated for selected plant species, and field measurement uncertainty sources are listed and discussed specifically for foliar pigments and canopy leaf area index. The review concludes with the main findings and suggestions as how to reduce uncertainties and include variability in scaling vegetation imaging spectroscopy signals and functional traits of single leaves up to observations of whole canopies.
- Keywords
- Imaging spectroscopy, Inversion, Multiscale validation, Quantitative remote sensing, Radiative transfer models, Retrieval of vegetation traits, Scaling, Uncertainty, Variability,
- Publication type
- Journal Article MeSH
Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use.
- Keywords
- Chloroplast counting, Norway spruce (Picea abies L. Karst.), confocal microscopy, coniferous needle structure, disector method, mesophyll, profile counting, stereology.,
- MeSH
- Models, Biological MeSH
- Chloroplasts metabolism MeSH
- Mesophyll Cells cytology metabolism MeSH
- Picea MeSH
- Imaging, Three-Dimensional methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The anatomical structure of mesophyll tissue in the leaf is tightly connected with many physiological processes in plants. One of the most important mesophyll parameters related to photosynthesis is the internal leaf surface area, i.e. the surface area of mesophyll cell walls exposed to intercellular spaces. An efficient design-based stereological method can be applied for estimation of this parameter, using software-randomized virtual fakir test probes in stacks of optical sections acquired by a confocal microscope within thick physical free-hand sections (i.e. acquired using a hand microtome), as we have shown in the case of fresh Norway spruce needles recently. However, for wider practical use in plant ecophysiology, a suitable form of sample storage and other possible technical constraints of this methodology need to be checked. We tested the effect of freezing conifer needles on their anatomical structure as well as the effect of possible deformations due to the cutting of unembedded material by a hand microtome, which can result in distortions of cutting surfaces. In the present study we found a higher proportion of intercellular spaces in mesophyll in regions near to the surface of a physical section, which means that the measurements should be restricted only to the middle region of the optical section series. On the other hand, the proportion of intercellular spaces in mesophyll as well as the internal needle surface density in mesophyll did not show significant difference between fresh and frozen needles; therefore, we conclude that freezing represents a suitable form of storage of sampled material for proposed stereological evaluation.
