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

Abstract

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.

Stereology, an Unbiased Methodological Approach to Study Plant Anatomy and Cytology: Past, Present and Future

“…Using specialized software, different virtual test probes with an arbitrary pre-defined (e.g., random) position and orientation can be generated within the stack of sections and can be applied directly to this 3D image data. Albrechtová et al (2007) presented the confocal stereological methods used to evaluate the mesophyll structure of narrow leaves, such as conifer needles. Kubínová et al (2014) showed the application of confocal microscopy for counting chloroplasts in a mesophyll cell using optical disector principle (see below).…”

“…Using this approach, the volume density of individual tissues in a leaf was measured by Edwards et al (1999), Klich (2000), Luković et al (2001), Bray and Reid (2002), Luković (2006), Marin et al (2006), Zorić et al (2011;, Moura and Alves (2014), Bernardo et al (2017), and Bertel et al (2017). The proportion of intercellular spaces in mesophyll was also investigated by Albrechtová and Kubínová (1991), Kukkola et al (2005), Albrechtová et al (2007), Lhotáková et al (2008), Psaras (2011), andZorić et al (2011;. The proportion of intercellular spaces in the palisade parenchyma was measured by Konoplyova et al (2008) from micrographs of paradermal sections of the leaves.…”

“…The proportion of intercellular spaces in the palisade parenchyma was measured by Konoplyova et al (2008) from micrographs of paradermal sections of the leaves. Furthermore, volume density of needle tissues (Albrechtová et al, 2007;Lhotáková et al, 2012), trichomes in the leaf (Marin et al, 2008), and petiole tissues (Luković et al, 2016) were estimated.…”

“…The Cavalieri principle, which is based on multiplying the sum of areas of SURS sections by the distance between the subsequent sections (Gundersen and Jensen, 1987), was used for estimating volume of leaf and/or leaf components by Kubínová (1989bKubínová ( , 1991Kubínová ( , 1993, Albrechtová and Kubínová (1991), Edwards et al (1999), Klich (2000), Marin et al (2008), and Albrechtová et al (2007). Detailed instructions on how to apply this method are given in the overview of stereological methods for the measurement of leaf characteristics (Kubínová, 1993).…”

“…1,2,8) are involved in the transfer of carbon dioxide during the process of light capture and photosynthesis, which has crucial importance for the function of the plant organism. Quantitative analysis of changes in the leaves can be very helpful in many applications studying the effect of environmental factors on plants, such as analysis of the effect of air pollution (Albrechtová et al, 2007), elevated CO 2 concentration (Lhotáková et al, 2012) or temperature (Juurola et al, 2005). Moreover, leaf geometrical characteristics may be useful for interpreting physiological measurements, three-dimensional (3D) modelling during photosynthesis (Juurola et al, 2005) and for phenotyping (Flood et al, 2016).…”