MicroscopyEducation


Table 1: Comparison of some stomatal features for the plants shown in Figures 3, 5, and 6.


Plant Agave


Masdevallia Peony


Stomatal density (mm−2


)


43 28 68


Stoma size (µm)


47×56 30×41 8.4×31


Results Stomatal density measurements. Figure 2b shows a low-


magnification view of a varnish replica used to compute the density of stomata on the underside of a leaf of the Blue Glow Agave plant similar to that shown in Figure 3. Each of the 232 dark spots is a stoma. Te leaf axis is vertical. Te area shown is 2.54 mm×2.13 mm=5.4 mm2 is 232/5.4=43 per mm2


, thus the density of stomata . In practice, stomatal densities are


determined using several varnish replicas on a leaf in order to obtain statistically valid results. Soſtware for this purpose is described in the literature [11]. Stereo images of the leaf surface. By combining the stack


of images taken at different depths, focus stacking enables cre- ation of stereo images. Tese images permit a detailed under- standing of the morphology of the leaf surface (Figure 4a). Such oblique stereo views show the structure of stomata more clearly than would be seen by looking straight down onto them. Measuring Individual Stomata. Te area of a stoma


opening is given by its width and length. Te width and length of the Agave stoma shown at the lower leſt of Figure 3 (middle image), and also in the center of Figure 4, were 56 μm and 47 μm, respectively. Te leaf axis is vertical in this view. Te depth and height of a stoma relative to a leaf surface was determined using the limited depth of field of a standard light microscope in conjunction with the microscope’s stage micrometer. In Figure 4b, the two zeroes at the top and leſt of the Agave oblique view are at the leaf surface. Te points marked -13μ lie 13 μm below the surface. Te points marked 8μ lie 8 μm above the leaf surface. Te cross-sectional view at the bottom of Figure 3 shows sections of two stomata, marked 1 and 2. Te A and B designations mark the locations of the depressions on either side of the center that are -13 μm deep in Figure 4. Figures 5 and 6 show plant images, planar views, and


cross-sectional views of stomata for a Masdevallia orchid and a Peony, respectively. Te cross-sectional views in Figures 3, 5, and 6 show that some stomata extend above the leaf surface, while others lie at or below the leaf surface. Table 1 shows a comparison of stomatal density and size


for the plants discussed here. Te numbers given in the table reflect measurements made on individual leaf areas and repre- sentative stomata. Tese numbers show significant variations in size and density of stomata from plants of different types. In the field, more measurements and statistical analyses are used to refine these numbers.


Discussion Tere is significant variability in size among stomata on different types of plants, and even on individual plants of a


16


particular species. Some stomata are simple, like those on the leaves of a Peony, while others are complex, like those on the leaves of an Agave. Some useful information about the mor- phology of stomata was obtained from single specimens, whereas information about a plant’s interaction with its envi- ronment would be best obtained from statistical analyses of many stomata. A web page with additional images and links to references


is available on the internet at www.paedia.com/Stomata.html. Photos of specimen preparation are also shown.


Conclusion Microscopic examination of stomata is one way we can


look for clues to help optimize the environment in which we live. In fact, these plant features make suitable subjects for classroom projects whereby students design experiments to determine the response of stomata to various environmental stimuli. Various


specimen preparations and observational


methods are used in studying stomata with light microscopy. In this article, focus stacks were used to obtain high depth-of- field images of leaf surfaces and their stomata. Te small depth of field typical of light microscopy without focus stacking was used to obtain measurements of stomata depth relative to the leaf surface. Te high-quality images of stomata shown in this article can be achieved by anyone with a monocular micro- scope, a digital camera, a personal computer, and focus-stack- ing soſtware.


Acknowledgment Special thanks are due to Brian Brown who built the stage microtome used in preparing cross sections of leaves.


References [1] Tere is a useful review of stomata on Wikipedia.com under the heading “Stoma.”


[2] JC McElwain and WG Chaloner, Ann Bot–London 76(4) (1995) 389–95.


[3] TJ Brodribb and SAM McAdam, PLoS One 8(11) (2013) e82057.


[4] T Lawson and MR Blatt, “Stomatal Size, Speed, and Responsiveness Impact on Photosynthesis and Water Use Efficiency”, Plant Physiology, American Society of Plant Biologists, April 2014.


[5] WRJ Van Cotthem, Bot J Linn Soc 63 (1970) 235–46. [6] V Fernandez and T Eichert, Crit Rev Plant Sci 28 (2009) 36–38.


[7] GE Gudesblat (2009) 1114–16.


et al., Plant Signaling and Behavior 12


[8] A Martins, “Enigma of the trees that resist wildfires.” BBC News. https://www.bbc.com/news/science-environ- ment-34116491) September 2015.


[9] HeliconSoſt Focus brand imaging soſtware, www.helicon- soſt.com.


[10] JF Eisele et al., PLoS One 11(10) (2016) e0164576. [11] KC Fetter et al., “StomataCounter: a deep learning method applied to automatic stomatal identification and counting,” bioRxiv.org, May 21, 2018, https://doi. org/10.1101/327494.


www.microscopy-today.com • 2019 January


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