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JXB Advance Access originally published online on February 24, 2007
Journal of Experimental Botany 2007 58(6):1451-1461; 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

Jana Albrechtová1,2,*, Jirí Janácek3, Zuzana Lhotáková1,3, Barbora Radochová3 and Lucie Kubínová3

1Charles University in Prague, Faculty of Science, Department of Plant Physiology, Vinicná 5, CZ-12844, Prague 2, Czech Republic
2Institute of Botany, Academy of Sciences of the Czech Republic, CZ-25246 Pruhonice, Czech Republic
3Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-14220, Prague 4, Czech Republic

* To whom correspondence should be addressed. E-mail: albrecht{at}natur.cuni.cz

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

Received 22 June 2006; Revised 18 December 2006 Accepted 22 December 2006


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