TITLE: Flavonol, Anthocyanin, and Chlorophyll Indices using a Force-A Dualiex Scientific+
AUTHORS: David LeBauer, Maria Newcomb, Matthew Herrit, Ebrahim Babaeian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols describes the use of a hand-held sensor to estimate polyphenols and cholorphyl content of leaves based on absorbance and transmitance of specific wavelenghts of visible and near infrared light.</div><div class = "text-block">The instrument does not directly measure these chemical concentrations, but measures reflectance and absorbance indices that have been demonstrated to relate to leaf level concentrations.</div><div class = "text-block">We use it to measure Chlorophyll, anthocyanin, and a nitrogen balance index.</div><div class = "text-block"><span>Chlorophyll Concentration (μg/cm</span><span style = "vertical-align:super;">2</span><span>): The instrument is factory calibrated to measure leaf chlorophyll concentration.</span></div><div style = "text-align :; float : ;"><span style = "">\textrm{Chl}=\frac{\textrm{NIR} - \textrm{Red}}{\textrm{Red}}</span></div><div class = "text-block">Flavanol Index (Flv)</div><div style = "text-align :; float : ;"><span style = "">\textrm{Flv} = \log \frac{\textrm{NIR fluo excited red}}{\textrm{NIR fluo excited UV-A}}</span></div><div class = "text-block">Anthocyanin Index</div><div style = "text-align :; float : ;"><span style = "">\textrm{Anth} = \log \frac{\textrm{NIR fluo excited red}}{\textrm{NIR fluo excited green}}</span></div><div class = "text-block">Nitrogen Balance Index (NBI): a relative measure of nitrogen status</div><div style = "text-align :; float : ;"><span style = "">\textrm{NBI}=\frac{\textrm{Flavanol Index}}{\textrm{Flavonol Index}}</span></div><div class = "text-block"><span>Note that hte instrument documentation does not provide specific wavelenghts, only approximate wavelengths in this diagram (from </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span>):</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/9ghdrvn.png" /></div></div>

[STEPS]
?. [Take measurements]
For each two row plot:For two subsamples per plot: Identify the 1-3 replicate plants per row to measure. For a standard 3 m row, this would be the plant closest to 1.5m from the end. For six subsamples / plot: Identify the center plant and two adjacent plants in each of the two rows
?. [Identify the youngest fully expanded leaf on each plant]
On each plant, the youngest, fully expanded leaf should be measured. To determine if a leaf is fully expanded, observe that the leaf is attached at the ligule; a leaf that is not fully expanded will still be partially wrapped around the stem.Image from Wikipedia CC-By 3.0 Kelvin Ma
?. [Identify a sunlit, horizontal location on each leaf to measure]
The measurement should be taken on a part of the leaf that is sunlitapproximately horizontal to the groundapproximately halfway between the leaf rib and edgefree of disease or herbivory.
?. [Attach Dualex and measure]
Clip Dualex sensor to leaf Click "OK"Instrument will collect measurements
?. [Record plot name and measurement number]
?. [Format and upload data    ]
See Data Entry documentation for BETYdb
?. [Take measurements]
Non-destructive measurements of biochemical pigments were obtained on the uppermost, fully expanded leaf The instrument calculates leaf chlorophyll index (Chl), flavonol index (Flv), and Nitrogen Balance Index (NBI). These measurements were taken from a plant within the sampling location.