The overall goal of this procedure is to determine the thermal properties of leaves in a contact-free manner. This method can help answer key questions in the field of molecular farming about in-process control of plant growth and health. The main advantages of this technique are that it is contact-free, fast, and does not damage the sample.
The implications of this technique extend toward automated inspection of plantations with a mobile detector to define feeding and nutrition on a plant-by-plant basis. To begin plant cultivation, flush mineral wool blocks with one to two liters of deionized water each, then flush each block with one liter of a 0.1%fertilizer solution. Place one tobacco seed in each block and flush with 250 milliliters of fertilizer solution, ensuring that the seed is not washed away.
Cultivate the plants for seven weeks in a greenhouse or phytotron. Once the plants have grown, either harvest intact, undamaged single leaves for plant characterization or use intact plants. To begin leaf thickness determination, prepare in autoclave 50 milliliters of a 2%mass by volume agarose solution and phosphate-buffered saline.
Allow the solution to cool to 40 degrees celsius and then add the solution to a Petri dish containing a single leaf sample. Cool the sample at four degrees celsius for 30 minutes to solidify the agarose and embed the leaf. Then use a vibratome to cut the agarose block into 200 micrometer slices.
Mount five transversal leaf sections on a glass slide with cyanoacrylate. Use a microscope or dial gauge to determine the thickness of vein-free areas of the leaf sample. Use another single leaf sample in water to determine leaf density with a pycnometer.
Next, clamp another single leaf in front of the detector in a UV vis spectrophotometer sample chamber. In the spectrophotometer software, select a spectrum of 900 to 1, 600 nanometers. Perform the scan and record the transmission value based on the spectral curve.
Then, clamp the leaf behind the detector and repeat the scan. Determine the reflection value from the spectral curve. Obtain transmission and reflection measurements from at least three leaf samples.
To begin, mount a 1, 550 nanometer fiber coupled single bar near-IR diode laser in the cone. Then secure a biconvex lens with a 25.4 millimeter focal length at the end of the cone. Clamp a 25.4 millimeter diameter cone with a stainless steel holder to a laboratory stand.
Place a neutral density filter with an optical density of 1.0 in a 22 millimeter thick ceramic plate above the censor to attenuate the laser beam. Mount a photo diode power sensor 354 millimeters below the bottom of the lens. Connect the photo diode power sensor to an oscilloscope.
Affix a 10 centimeter by 10 centimeter frame with a six centimeter by six centimeter sample exposure area 308 millimeters below the lens. Mount the detector 135 millimeters above the ceramic attenuator. Connect a near-IR detector to a computer and prepare the software.
Put an absorber plate on the leaf and turn on the laser. Adjust the detector angle to 45 degrees relative to the laser beam. Continue adjusting the detector angle and height until the maximum temperature reading is observed.
In the laser control software, select power control. Set the output laser power to five watts and the pulse duration to 0.5 seconds. To begin the measurements, either bring a whole plant to the measurement apparatus or harvest whole, undamaged single leaves.
Immediately mount the leaf sample in the frame, taking care not to damage the leaf. Do not allow the leaf to contact the ceramic attenuator. In the near-IR software, start a new 60 second measurement.
Record the baseline temperature profile for 10 seconds before activating a single 0.5 second laser pulse. Continue recording data for the remaining 49.5 seconds. When the measurement is complete, click on the stop button above the thermal profile and then save the profile.
Export the raw time and temperature data as a dot dat file for further analysis. Then in the oscilloscope software, determine the flank heights in the voltage profile, which were automatically collected during the laser pulse. Repeat the temperature profile measurement with a single 0.5 second laser pulse for each sample.
Then repeat the laser signal measurement without a leaf sample as a reference. Determine the flank heights from this voltage profile and calculate the transmission values for each sample. Use spreadsheet software to calculate and graph the specific heat capacity and thermal conductivity of the samples.
Single leaf samples of the plant species Nicotiana tabacum, known as tobacco, and Nicotiana benthamiana were analyzed with this method. Both species showed a rapid increase to maximum temperature in less than one second in response to the laser pulse, followed by an exponential decrease to ambient temperature. The specific heat capacity and thermal conductivity values were calculated for both species from leaves collected at the bottom, middle, and top of the plant.
An increase in thermal conductivity for leaves further up the plant was observed for both species, however Nicotiana tabacum showed an inverse trend in specific heat capacity. No correlation between cultivation duration and thermal conductivity was observed in Nicotiana tabacum. Further, no correlation between cultivation conditions and thermal properties were observed in Nicotiana benthamiana.
The effect of individual variables on specific heat capacity and thermal conductivity was examined. The thermal conductivity of Nicotiana benthamiana was highly sensitive to ambient temperature. Specific heat capacity was not significantly sensitive to any of the parameters involved in its calculation.
Once mastered, this technique can be done in a few minutes per sample if it is performed properly. While attempting this procedure, it is important to remember to correctly mount the leaf at the given precision to ensure high quality measurements and reproducible results. Following this procedure, other methods like fluorescence measurements can be performed in order to answer additional questions like recombinant protein expression.
After its development, this technique paved the way for researchers in the field of plant biotechnology to explore plant growth in Nicotiana species. After watching this video, you should have a good understanding of how to determine the specific heat capacity and thermal conductivity of leaves in a contact-free and non-destructive manner. Don't forget that working with lasers can be hazardous to your eyes and precautions such as wearing the appropriate safety glasses should always be taken while performing this procedure.
