title: Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach.
abstract: 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.
authors: Charles University in Prague, Faculty of Science, Department of Experimental Plant Biology, Viničná 5, 128 44 Prague 2, Czech Republic.
authors: Zuzana
authors: Z
authors: Kubínová
authors: Jirí
authors: J
authors: Janácek
authors: Zuzana
authors: Z
authors: Lhotáková
authors: Lucie
authors: L
authors: Kubínová
authors: Jana
authors: J
authors: Albrechtová
mesh_terms: D002736:Chloroplasts / Q000378:metabolism*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D058503:Mesophyll Cells / Q000166:cytology* / Q000378:metabolism; D008954:Models, Biological; D028222:Picea
keywords: Chloroplast counting; Norway spruce (Picea abies L. Karst.); confocal microscopy; coniferous needle structure; disector method; mesophyll; profile counting; stereology.