Stimulation and Observation of Leaf Stomata


observed as they change phase and composition with changes in temperature, ambient gas levels, pressure, and illumination.


Conclusions An environmental chamber used in conjunc-


tion with a light microscope permitted observa- tion of the opening and closing of leaf stomata in two controlled experiments. Stomatal openings and closings were observed and measured as first humidity and CO2


concentration, and then light


Figure 6: Experiment No. 1. Changes in T. spathacea stomatal open area prompted by two events. Prior to Event #1 leaf was exposed to room air containing nominally 800 ppm of CO2


Event #1 scrubber was connected reducing CO2 level to zero and RH=85%. At Event #2 the scrubber was disconnected, again exposing the leaf to 800 ppm of CO2 and RH=65%.


and RH=65%. At


were varied within the chamber. Transient and equilibrium responses to predetermined environ- mental conditions were observed. Detailed still and moving picture representations of stomata were obtained using focus-stacking photogra- phy. Although leaf stomata are featured here, the apparatus and methods described could be used to study many other subjects.


Acknowledgment Special thanks are due to Brian Brown who


helped make the apparatus. Patent protection is pending for several aspects of the apparatus described herein.


References [1] D Clark, Microscopy Today 27(1) 12–16.


(2019)


[2] Z Sun et al., PLOS Comput Biol, 10(3) (2014) 1003930.


[3] LI-COR, Inc., “LI-6800 Portable Photo- synthesis System.” https://www.licor.com/ env/products/photosynthesis/LI-6800 (accessed April 30, 2019).


Figure 7: Experiment No. 2. Changes in T. spathacea stomal opening extent in response to two events. Prior to Event #1 stomal opening equilibrated at 5 μm in red light of intensity 300 μmol/ m2


·sec. At Event #1 blue light of intensity 100 μmol/m2 established. At Event #2 the blue light was switched OFF, and the opening returned to 5 µm.


the maximum opening because the increase in size stopped abruptly. At Event #2, 91 minutes from the start of the experi- ment, the B light was switched OFF. Te stoma began closing immediately and exhibited a slight undershoot followed by an overshoot before returning to a final equilibrium value of 5 μm aſter 174 minutes.


Discussion Te apparatus shown here enables the macroscopic and


microscopic study and observation of plant stomata in a con- trolled environment. In further experiments, gases entering and leaving the chamber could be compared for the study of transpiration and respiration while simultaneously observ- ing individual stomata as they open and close. Other living organisms such as algae, molds, bacteria, insects, and the like could also be studied under the variety of conditions achiev- able within the chamber. Non-biological specimens could be


2019 July • www.microscopy-today.com


[4] Helicon Soſt Ltd, “Helicon Focus.” http:// www.heliconsoſt.com (accessed April 30, 2019).


·sec was added, and a new equilibrium was


[5] LED light source model LZP-00MD00 from LED Engin of San Jose, CA.


[6] D Clark and B Brown, Microscopy Today 24(4) (2016) 12–15.


[7] Analog Devices, Inc., “Optimizing Precision Photodiode Sensor Circuit Design.” https://www.analog.com/en/tech- nical-articles/optimizing-precision-photodiode-sensor- circuit-design.html (accessed April 30, 2019).


[8] Jorgensen Labs, “JORVET Soda Lime, 3 Lb.” https://www. jorvet.com/product/jorvet-soda-lime-3-lb (accessed April 30, 2019).


[9] CO2


sensor model COZIR-LP CO2 Sensor from Gas Sens- ing Solutions of Cumbernauld, Scotland.


[10] Humidity sensor model HIH-4030 from Honeywell Inter- national Inc. of Minneapolis, MD.


[11] S Assmann, Plant Physiol 87(1) (1988) 226–31. [12] TD Sharkey and K Raschke, Plant Physiol 68(5) (1981) 1170–74.


[13] Photoshop Elements by Adobe.com. 23


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