Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

Journal of Experimental Botany
Zuzana KubínováJana Albrechtová

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 mesoph...Continue Reading

References

Sep 1, 1987·Journal of Microscopy·H J Gundersen, E B Jensen
Jul 27, 1999·The Plant Journal : for Cell and Molecular Biology·J L MarrisonR M Leech
Sep 1, 1979·Plant Physiology·S A BoffeyR M Leech
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Jan 26, 2013·Journal of Experimental Botany·Shunsuke AdachiTadashi Hirasawa
Apr 1, 1968·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·N O Frandsen

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Citations

Apr 3, 2014·Plant, Cell & Environment·Matt StataRowan F Sage
Mar 7, 2015·Cell and Tissue Research·Lucie Kubínová, Jiří Janáček
Sep 3, 2016·Frontiers in Cell and Developmental Biology·Logan W Cole
Feb 23, 2019·Plant Molecular Biology·Kanagesswari MuniandySadequr Rahman
May 5, 2017·Journal of Experimental Botany·Jayakumar BoseStephen D Tyerman
Aug 28, 2021·Cells·Abdul HameedBrent L Nielsen

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Methods Mentioned

BETA
light microscopy
confocal microscopy

Software Mentioned

Cortona
NCSS
IRIS Explorer
Cortona 3D
Ellipse Point Grid
Ellipse

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