Stimulation and Observation of Leaf Stomata Using a Light Microscope
Douglas Clark Paedia LLC, 15 Conrad St., San Francisco, CA 94131-2924
dclark@paedia.com
Abstract: Individual plant stomata were studied within a sealed environmental chamber that encloses a volume of space around a microscope objective. A leaf that is attached to a plant is secured to a temperature-controlled microscope stage, and the top surface of the leaf is illuminated via the microscope’s light source while stomata on the underside of the leaf are observed through a microscope objec- tive. Stomatal responses are observed on a leaf exposed to envi- ronmental conditions including various illuminations and changes in ambient CO2
level, at a given temperature.
Keywords: Stomata, plants, quantitative measurements, light micros- copy, environmental chamber
Introduction In a previous article [1], I presented detailed images of sto-
mata on the leaves of various plants. Tat article discussed the importance of understanding the properties and functions of stomata in a number of fields as they relate to understanding living conditions on Earth. Two parameters discussed in that article were the size and density of stomata on the undersides of leaves. Te measure of stomatal size was expanded to include the heights and depths of various features above and below a leaf ’s surface. In that previous work, obtaining stomata to study at various degrees of openness was a challenge. Photographic study of stomata is helped by
understanding the environmental conditions nec- essary to control their opening and closing. Put simply, when water enters a stoma’s guard cells they become turgid and the stoma opens. When water leaves the guard cells, the stoma closes. Te mechanisms behind these actions are complex. A network of 70 components is involved in stomatal opening in response to levels of CO2
, red light, blue
light, and ABA (abscisic acid), a plant hormone [2]. At a macroscopic scale, transpiration and res-
piration during photosynthesis are studied using commercially available equipment [3] that mea- sures the conductance of CO2
and water vapor
through a large number of stomata. To observe the opening and closing of one or a few stomata at a microscopic scale, it would be useful to observe a living leaf within a sealed test chamber on a micro- scope and expose the leaf to a combination of envi- ronmental variables such as gases, temperature, humidity, and light. With a large enough specimen, differences in gas concentrations that enter and leave the chamber could be measured and related to photosynthesis within the leaf. With a large leaf area contained inside the chamber, many stomata would be involved in such a measurement, yet a user could observe individual stomata using the microscope.
18 In this article, I describe such an environmental cham-
ber and related equipment for use with a light microscope. Te chamber exposes a section of a living leaf (that is, a leaf attached to a plant) to various levels of stimuli including light, gas flow, and temperature. Adjusting these variables can stim- ulate the opening and closing of stomata of leaves in vivo.
Materials and Methods Te experimental setup (Figure 1) comprises a microscope
with a light source, a sealed environmental chamber, a micro- scope stage, a source of gas, a thermal source, and sensors for measuring light intensity, humidity, temperature, and CO2
lev-
els. Excepting the microscope, most of the components were obtained online. Microscope. A Nikon Eclipse LV100 microscope and a
Point Grey Model GS3-U3-51S5C-C digital camera were used in this work. Te camera was mounted on a 0.65× relay lens in the microscope’s trinocular port. Instead of a standard micro- scope, an inverted microscope can be used by inverting the setup shown in Figure 1. Snapshot images were obtained using soſtware provided by the camera manufacturer. Focus-stack- ing soſtware was used to increase the depth of field of most
Figure 1: An experimental microscope setup showing a chamber for isolating a living leaf and exposing it to various environmental conditions. The stippled regions indicate where the objective lens and the leaf are sealed off from the atmosphere.
doi:10.1017/S1551929519000622
www.microscopy-today.com • 2019 July
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