Photography of Plant Stomata




Still and Time-Lapse Photography of Plant Stomata

Elkins C. B., Williams G. G. (1962)

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Control of Leaf Stomatal Opening

by Johnson R. (2007)

Russell Johnson

in Colby J. Res. Meth. 9: 14-17 –

The opening and closing of stomata is a very important mechanism that plants use to control the diffusion of gases in and out of leaves. Ideally stomata must be sufficiently open to allow enough CO2 (needed for photosynthesis) to diffuse in, but sufficiently closed to prevent too much evaporative loss of H2O. This is sometimes a difficult balance to achieve and the amount of stomatal opening is controlled by a large number of factors. The degree to which stomata are open can be observed and measured by making imprints of the leaf epidermis and viewing (and photographing) these imprints using a light microscope. Once this technique is mastered it can then be used to measure the effects on stomatal aperture of a wide variety of environmental and chemical factors. Epidermal imprints can be made by applying a dental resin to the surface of the leaf and allowing it to harden. This negative impression can be archived and used to make a positive image (with clear nail polish) whenever desired.


I. Procedure for making imprints of the leaf epidermis


II. Procedure for making digital records (movie clips) of epidermal surfaces.


III. Procedure for measuring stomatal aperture from digital images.



Analysis of stomatal apertures with minimal leaf manipulation

Fig 1. Experimental setup for stomatal aperture measurements. (a) Schematic representation of the workflow; (b) epifluorescent microscopic picture of the Arabidopsis leaf stained with rhodamine 6G; (c) the same picture as in (b) after application of the option “sharpen” in ImageJ. Bars, 50 μm. –


A Rapid and Simple Method for Microscopy-Based Stomata Analyses

Eisele J. F., Fässler F., Bürgel P. F., Chaban C. (2016) 

Jochen F. Eisele, Florian Fässler, Patrick F. Bürgel, Christina Chaban

Department of Plant Physiology, Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany


in PLoS ONE 11(10): e0164576. doi:10.1371/journal. pone.0164576

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Fig 3. Visualization of stomatal apertures in intact leaves and epidermis peels. (a,b) Epifluorescent images of intact leaves mounted in water (upper panel) and in 30% glycerol (lower panel). (a) Staining with 1 μM rhodamine 6G; (b) staining with 10 μM rhodamine 6G. (c) Photograph of leaf epidermis peels. (d) Confocal image of cells in the peeled epidermis; λexc = 488 nm, λem = 505–545 nm (upper panel); λexc = 561 nm, λem = 600–640 nm (middle panel); bright field (lower panel). Bars, 50 μm. –


There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures.

Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required.

Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results.

We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli.

We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis.

The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.

Method of making epidermal imprints



An improved method of making epidermal imprints

by Horanic G. E., Gardner F. E. (1967)


F. E. Gardner

in Bot. Gaz. 128: 144-150 – –


A rapid and simple way to make imprints of plant epidermal structure for microscopic study is described. Rhoplex AC-33, a viscous emulsion of acrylic polymers, is applied to the tissue, allowed to dry to a transparent film, then peeled off, and placed on a microscope slide.

The film records excellent structural detail and is relatively permanent. Rhoplex is non-toxic to the plant, and repeated imprints can be made for studies of guard cell movement or of epidermal cell development.

Photomicrographs of various epidermi are presented.