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.

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This review presents an historical overview of stereological methods used for the quantitative evaluation of plant anatomical and cytological structures. It includes the origins of these methods up to the most recent developments such as the application of stereology based on 3D images. We focus especially on leaf, as the vast majority of studies of plant microscopic structure examine this organ. An overview of plant cell ultrastructure measurements as well as plant anatomical characteristics (e.g., plant tissue volume density, internal leaf surface area, number and mean size of mesophyll cells and chloroplast number), which were estimated by stereological methods most frequently, is presented. We emphasize the importance of proper sampling needed for unbiased measurements. Furthermore, we mention other methods used for plant morphometric studies and briefly discuss their relevance, precision, unbiasedness and efficiency in comparison with unbiased stereology. Finally, we discuss reasons for the sparse use of stereology in plant anatomy and consider the future of stereology in plant research.

This chapter gives examples of basic procedures of quantification of plant structures with the use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.

The main objective of this study was to find out whether the selected chloroplast characteristics measured in the mesophyll layer nearest to the needle surface (i.e., the first mesophyll layer) could be representative for the whole needle cross section. Two chloroplast sampling approaches were applied on Norway spruce needles during the investigation of the effects of different levels of air CO2 concentration and irradiance: (i) sampling only from the first mesophyll layer, and (ii) systematic uniform random (SUR) sampling. The selected characteristics were: (i) chloroplast area, (ii) starch grain area, and (iii) starch areal density on median chloroplast cross sections, and (iv) chloroplast number per unit of needle volume. It was shown that the first mesophyll layer was not representative for estimating all evaluated characteristics except the chloroplast area. Sampling only there caused obtaining slightly biased results, while SUR sampling gave unbiased estimations at the cost of longer measuring time. The major effect of studied factors was in starch areal density and starch grain area, which were larger in sun needles in elevated CO2 concentration in comparison with sun needles in ambient CO2 concentration. In conclusion, it was demonstrated that the first layer of mesophyll is not always representative for the needle cross section. If technically feasible, SUR is recommended for analysis of chloroplast ultrastructure. The simplified sampling design can be applied, e.g., for comparisons of many different treatments. However, it should be combined with other approaches to characterize the chloroplast function and the results carefully considered and interpreted.