Stimulation and Observation of Leaf Stomata
images [4]. Both soſtware applications run on a Windows 10 personal computer. Light sources. For small-area illumination, an LED light
source [5] replaced the incandescent bulb in the microscope’s lamp house below the condenser (Figure 1). Te source sup- plies red (R), green (G), blue (B), and white (W) light. An asso- ciated power supply energizes each color independently. Te LED source is mounted on a water-cooled heat sink to permit high-power operation. Te R, G, and B wavelengths emitted by the source are nominally 623, 525, and 456 nm, respectively. Te W color temperature is 6500 deg. K. Te W light source is used when stomata are being photographed. Koehler illumina- tion was used for photography in most cases. For large-area illumination, the microscope’s condenser
above the incandescent light source was defocused, that is, lowered by a predetermined amount. Tis results in a larger, unfocused source of light that illuminates a larger area of a leaf. In a variant of this arrangement, the microscope’s con- denser is removed and collimated light from the microscope’s light source impinges on a leaf, resulting in a larger illuminated area. In yet another arrangement, an LED light source replaces the microscope’s condenser and illuminates a leaf specimen directly. Tese arrangements can be used when it is desired to irradiate a large portion of a leaf in order to measure gas exchange while observing stomata. Instead of using LEDs, a Diabloc Multifilter [6] can be
placed between the microscope’s incandescent light source and the condenser. White light illuminates the multifilter, which then is adjusted to simultaneously pass red and blue light of predetermined intensities. Light source calibration. Calibration of the light intensity
applied to a leaf was accomplished at the position of the leaf sur- face. In the example experiments described below, R and B light were used to stimulate a leaf. A photodetector was used to cali- brate the intensity of each color. A widely used unit for express- ing photosynthetically active radiation (PAR) intensity is moles of photons/area·time. For convenience in representing a large number of anything, Avogadro’s number may be used. In the present case, a mole=6.02×1023 m2
photons. Values of 100 μmol/ when CO2
·sec for each color are sufficient to cause most stomata to open levels are low. For calibration, the microscope’s objec-
tive lens was moved aside and a photodetector (1 in Figure 2) was placed in the beam of light passing through the microscope’s stage at a point where a leaf’s surface will reside. An electronic circuit was used to measure the photodetector current. Te light system’s controls were then calibrated in μmol/m2
·sec. Tis con-
cept is well-known and is discussed elsewhere [7]. Gas source. In the experiments described below, two con- were applied to a leaf being studied. In a first
centrations of CO2
case, ambient room air was supplied to the chamber by direct connection to an aquarium air pump with a pumping capacity of 2 liters/min. Te nominal concentration of CO2
in the author’s
laboratory air was 800 ppm, and the relative humidity was 65%. Te CO2
400 ppm; however, it is not uncommon for the CO2
concentration in outdoor air was measured to be about level in a
closed space to reach 700–800 ppm when people are present. In
a second case, air with a below-ambient concentration of CO2 was supplied by passing air from the aquarium pump through a cartridge containing high moisture content (12–19%) granulated
20
Figure 3: Chamber that seals the leaf from the atmosphere, shown here with the lower foam gasket that seals the leaf (sealing material for the objective lens is not shown).
www.microscopy-today.com • 2019 July
Figure 2: Experimental setup: photodetector (1), thermocouple (2), and heat- ing element (3).
soda lime [8]. In this case, the CO2 concentration was 0 ppm,
and the relative humidity was 82%. Gas entering the chamber was analyzed using commercially available sensors [9,10]. In the simple experiments described below, gas leaving the chamber was not evaluated. Effects related to other gases and even pres- sures could be investigated with this system. Chamber. A chamber (Figure 3) was made from a piece of
rigid plastic tubing, sized to fit freely over a microscope objec- tive. Gaskets seal the gaps between the tubing and the objective, as well as the tubing and the leaf, on the microscope stage. Te gaskets allow sliding movement of the chamber at each sealing
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
