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Stimulation and Observation of Leaf Stomata


location. Gas enters and leaves the chamber via two hose barbs. A resistive heating element (3 in Figure 2) was affixed to the underside of the microscope’s stage in order to set the tempera- ture of a leaf specimen. A thermocouple (2 in Figure 2) was secured to the microscope stage as close as possible to the leaf. It could also be placed in contact with the leaf itself. Gas analysis. For a small-area illumination, as described


above, only a small area of a leaf was illuminated, affecting only a small number of stomata, and differences between gas entering and leaving the chamber were small. In the large-area illumina- tion experiments, there would be sufficient concentration dif- ferences between the gas entering and leaving the chamber that the gas could be analyzed using commercially available sensors. Differences between entering and exiting gas levels would be a measure of the level of the photosynthesis taking place. In the present experiments, no gas analysis was undertaken. Plant leaf intact. Aſter an experiment was completed,


the leaf was easily removed from the microscope stage and leſt intact on the plant being studied.


Results Two experiments are described below. Both demonstrate


transient openings and closings of a single stoma in response to a step change in variables. A Tradescantia spathacea leaf was secured to a microscope stage by masking tape (Figure 4a). Te leaf was oriented with its stomata facing upward, that is, toward the objective lens. As a starting point for these experi- ments, the temperature, light levels, and wavelengths used in the study of stomatal opening were near those reported in the literature [11,12]. Te effects of abscisic acid levels were not studied in these experiments. Experiment No. 1. In this experiment, temperature and


illumination were held constant while the CO2 concentra-


tion and humidity in the chamber were varied between two extremes. Te following steps were undertaken:


1. Koehler illumination was used. 2. R and B light LED light sources were activated, and both were set to deliver 100 μmol/m2 microscope stage.


·sec at the surface of the


3. An attached leaf on a recently irrigated T. spathacea plant was taped across the microscope stage with the underside of the leaf facing upward, toward the objective lens.


4. Te heater attached to the stage was set to a temperature of 25 °C.


5. Te chamber was placed over the a 50× objective lens and slid downward so that its lower gasket formed a seal against the leaf (Figure 4b).


6. A hose was connected from an air source to the inlet hose connector on the chamber. Room air with RH=65% and a CO2


for 40 minutes. Room air could also be pumped through a CO2


soda lime [8], then through the chamber. Tus, the con- centration of CO2


varied between zero ppm (directly from the scrubber) and 800 ppm with the scrubber removed. Relative humidity of the air entering the chamber was 82% with the scrubber present and 64% with the scrubber absent.


2019 July • www.microscopy-today.com


level of 800 ppm was pumped through the chamber scrubber consisting of a tubular column filled with in air passing through the chamber was


Figure 4: Setup for observation of Tradescantia spathacea. (a) Securing a live leaf to the microscope stage with tape. (b) Chamber installed over leaf on microscope stage: chamber (1), chamber-objective gasket (2), chamber-stage gasket (3), and hose from gas source (4).


7. A stoma of interest was selected for observation. Te R and B lights were temporarily switched off, and the W light was energized long enough to record image stacks of the stoma. Aſter picture-taking was done, the W light was switched off and the R and B lights were turned back on. Tis procedure was done at 10-minute intervals.


experiment the stoma was stimulated to open and close by changing the humidity and concentration of CO2


Results from Experiment No. 1. During the example in the cham-


ber. Figures 5a and 5b show a T. spathacea stoma in open and closed conditions, respectively. Imaging soſtware [13] was used to measure the area of the stomatal opening. A set of images used for areal measurement was 2048 pixels wide. Each square pixel had an area of 10−2


μm2 . In each image, the area within


the open stoma was determined manually using the “lasso” feature of the imaging soſtware. A histogram function within the soſtware then gave the number of pixels within the area. Tat number multiplied by the area of a pixel gave the area of the open stoma. Te area of the stomatal opening was recorded over time


as the leaf was stimulated by two events. Te results are shown in Figure 6. Te abscissa is elapsed time in minutes, and the ordinate is the area of the stomatal opening in μm2


. Prior to the 21


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