## 2 Object Fields and Methods
### Diagnosis of Fruit Tree Vigor Using Optical Sensing
#### 2.3.2 Vigor Measurement of Mandarin Orange Leaves by Fluorescence and Fluorescence  
Two types of leaves of which the vigor greatly differed in the same cultivation environment were selected from the orange grove. Ten leaves were taken from three weak vigor trees and another 10 leaves were taken from three trees with strong vigor (Figure 4). These leaves were taken from shoots without fruits. The evaluation criteria of tree vigor are based on the tree size, the number of leaves per tree, and the leaf color. The collected leaves were sent cool to the laboratory at Mie University using a courier and measurements by fluorescence and fluorescence X-ray methods were performed in the laboratory. As for the fluorescent measurement, a Dualex Scientific+ instrument (Force-A, Orsay, France) was used. It provided indices of the flavonoids (FLAV), anthocyanins (ANTH), and chlorophyll (CHL) [14]. The leaf chlorophyll content was assessed by measuring the light transmission at 710 nm, absorbed by chlorophyll, and in the near-infrared (NIR) at 850 nm to take into account the effects of the leaf structure. The chlorophyll Dulex index is given by the formula:  
\[\mathrm{CHL}=[(\mathrm{I_{850}}/\mathrm{I_{0,850}})/(\mathrm{I_{710}}/\mathrm{ I_{0,710}})]-1 \tag{1}\]  
where I and I\({}_{0}\) are the signals measured with and without the leaf sample in the leaf clip, respectively. The Dualex (Dx) measures the leaf epidermal flavonoids or anthocyanins at 375 and 520 nm, respectively, using the chlorophyll fluorescence (ChIF) screening method and equalizing the ChlF signals under these excitation wavelengths and that under a red excitation at 650 nm as a reference. Compounds present in the epidermis of the leaves attenuate the incident radiation before this can reach the first chlorophyll layer present in the mesophyll, depending on their absorption spectrum. Flavonols are the main flavonoids in dicotyledons absorbing UV radiation at 375 nm; therefore, the intensity of the ChlF induced by this radiation (ChIF_UV) will be inversely proportional to the epidermal flavonoid concentration. Using a red light excitation, not attenuated by flavonoids, a ChlF signal (ChIF_R) independent of the flavonoid concentration is obtained. This signal is used as a reference. By comparing the ChlF signals from the two different excitations, the index of the flavonoids can be calculated (in accordance with the Beer-Lambert law) as the logarithm of the ratio between the ChlF under red light and that under UV radiation:  
\[\mathrm{FLAV}=\mathrm{log}(\mathrm{ChIF\_R}/\mathrm{ChIF\_UV}) \tag{2}\]  
The same concept applies to the determination of anthocyanins using a green light, absorbed by anthocyanins, instead of UV radiation. In addition to the above indices, the Dx sensor calculates the nitrogen balance index (NBI). \[\mathrm{NBI}=\mathrm{CHL}/\mathrm{FLAV} \tag{3}\]  
Figure 4: Sampled leaves: (**a**) Leaves of strong tree; (**b**) Leaves of weak tree. as the ratio between the chlorophyll and flavonol indices that can be used as a proxy of the crop leaf nitrogen level. Fluorometric analysis with the measurements of the chlorophyll, flavonoid and anthocyanin Dulaex indices (CHL, FLAV, ANTH) was performed using 10 leaves of good color and another 10 leaves of bad color. The measurements were made at three areas of each leaf shown in Figure 5 in order to find the pigment distribution inside the leaf. As for the fluorescence X-ray measurement, an Innov-X DELTA Premium Handheld XRF (hXRF) Analyzer (Olympus Corp., Tokyo, Japan) was used [15]. Although this hXRF sensor is portable, the specification of this sensor is as good as that of the Rayny EDX-700 (Shimadzu Corporation, Kyoto, Japan) for the laboratory use that was used in previous study [16]. Weighing roughly 2 kg, the instrument is equipped with a built-in camera/collimator mounted in the vicinity of the probe and a rechargeable Li-ion battery for easy field operation.