1. A New Method for Spectroscopically Estimating Leaf Nitrogen Concentration Zachary J. Bortolot CEARS

2. Acknowledgements Dr. Randolph H. Wynne Virginia Tech Conservation Management Institute U. S. National Guard NCASI McIntire-Stennis Research Program NASA International Society of Arboriculture Research Trust

3. Why Foliar Nitrogen? Fertilization – Nitrogen is a macronutrient. In order for a plant to grow and be productive, it must be able to extract large quantities of nitrogen from the soil. – However, too much nitrogen can be detrimental to plant growth and productivity, and the excess may leach into streams and lakes, causing environmental degradation. – Managers use foliar nitrogen measurements to determine whether nitrogen needs to be added, and if so how much.

4. Why Foliar Nitrogen? (cont’d) Forest Ecology – The carbon to nitrogen ratio in leaf litter determines how quickly the litter will decompose, and the quantify and chemical form of the nutrients after release. – This is important for monitoring the nitrogen and carbon cycles within a forest. – Foliar nitrogen is also very useful in determining a tree’s photosynthetic rate.

5. Chemical Determination of Foliar Nitrogen In the past, most managers have determined foliar nitrogen by collecting leaf samples from a plant and then sending the sample to a laboratory for analysis. Although the results are very accurate, there are limitations: – Cost – Timeliness – Sample size – Sample collection

6. Alternatives To solve these problems, several alternatives have been found. The Cardy ion meter – Involves inserting electrodes into the petiole (stalk) of a leaf to measure the nitrate concentration. – Inexpensive and quick, but the results are poor. Sample size and collection may still be a problem.

7. Alternatives (cont’d) Chlorophyll (a.k.a. SPAD) meters – Transmit light through a leaf to measure the leaf chlorophyll concentration in terms of ‘SPAD units’. – Nitrogen concentration is correlated to SPAD units because nitrogen is a major building block of chlorophyll. – This technique is quick, cheap and works well for a single species on a single site at a single point in time. – However, sample collection may be difficult and samples must be sent to a lab to obtain calibration coefficients.

8. The Use of Reflectance Spectroscopy Reflectance spectroscopy involves using the fraction of light reflected by an object to find out about the object.

9. Advantages of Reflectance Spectroscopy You do not need to physically be in contact with the leaves. It is possible to sample many leaves at the same time (may or may not be an advantage). If you use an airborne sensor, you may be able obtain a complete sample. If a ground based collection method is used, it is also easy to obtain many samples since the procedure is quite fast. The results are usually very accurate.

10. Disadvantages of Reflectance Spectroscopy Cost – The current cost of collecting the type of airborne data needed for these measurements make it infeasible for most applications. – Handheld units involve a high one-time cost ($20,000+) but future costs are quite small. In the past, the techniques used to determine foliar nitrogen yielded equations that were site and species dependent, or required that samples be dried and ground prior to measurement.

11. A Bit of Spectroscopic Theory Objects reflect different amounts of light at different wavelengths. A plot showing the amount of light reflected at different wavelengths is known as a spectral reflectance curve.

12. Spectral Reflectance Curves

13. Spectral Features The patterns seen in spectral reflectance curves are a result the chemistry and structure of the object being measured. For measuring foliar nitrogen levels, we are most interested in a type of feature known as vibrational absorptions.

14. Vibrational Absorptions Vibrational absorptions occur when light causes molecules or parts of molecules to vibrate. This happens at certain critical wavelengths. Which wavelengths these absorptions occur at is dependent on the structure on the molecule.

15. Example: Water

16. Vibrational Absorptions of Organic Materials A = Modern materials B = Ancient materials 1 = OH 2 = NH 3 = CH 4 = Organic pigments (not vibrational)

17. Absorption vs. Reflectance An important concept in spectroscopy is that of reflectance versus absorption. Reflectance is the fraction of light at a given wavelength that strikes the surface and then returns from the surface at the same wavelength.

18. Absorption vs. Reflectance (cont’d) Reflectance is not an ideal means of measurement in spectroscopy. This is because the decrease in reflectance due to an absorption is not proportional to the amount of the absorbing material present.

19. Absorption vs. Reflectance (cont’d) Example: – You add an amount of a material that absorbs 50% of the incident light to a bright surface (reflectance = 0.80) and to a dull surface (reflectance = 0.20). – Case one The reflectance decreases from 0.80 to 0.40, a difference of 0.40. – Case two The reflectance decreases from 0.20 to 0.10, a decrease of 0.10. – Even though the same amount of material is present, the reflectance decreases different amounts.

20. Absorption vs. Reflectance (cont’d) To fix this problem, we convert from reflectance to absorption using Beer’s law: – A = log 10 (1 / R) A = Absorption R = Reflectance – Going back to the example: Case 1: R = 0.80  A = 0.097; R = 0.40  A = 0.40; Change in A = 0.30 Case 2: R = 0.20  A = 0.70; R = 0.10  A = 1; Change in A = 0.30

21. Foliar Nitrogen For measuring foliar nitrogen, there are a number of relevant absorption features. Of particular interest are two vibrational absorptions caused by proteins that are closely related to nitrogen concentration. Unfortunately, there are absorptions due to other molecules at the same wavelengths as these two absorptions.

22. The Absorption of Interest The Absorptions Together with its Neighbors The Absorptions on their Own

23. Leaf Water 40 – 80% of a green leaf’s mass is water. Unfortunately, the leaf water has an dramatic affect on the reflectance at the wavelengths where the absorptions we are looking at occur.

24. The Affect of Leaf Water

25. Methods for Determining Leaf Nitrogen: The Derivative Method In the past, several approaches have been used to predict leaf nitrogen concentration from spectroscopic data. The original and most common method is known as the derivative method. The first step is to convert leaf spectrum from reflectance to absorption. Generally the entire leaf spectrum is used, not just spectral regions known to contain useful features. Next you subtract the absorption at each wavelength at from the absorption at the previous wavelength. The result is known as a first difference spectrum.

26. Methods for Determining Leaf Nitrogen: The Derivative Method (cont’d) Stepwise multiple linear regression is then used to develop a model relating the values in the first difference spectrum to foliar nitrogen concentration. This method works because it is able to pick up the subtle changes in curvature caused by vibrational absorptions related to nitrogen.

27. Methods for Determining Leaf Nitrogen: The Derivative Method (cont’d) Note the changes in curvature at different N concentrations.

28. Methods for Determining Leaf Nitrogen: The Derivative Method (cont’d) However, this method has limitations: – A large number of wavelengths are typically used, necessitating a large number of training samples. – The models often include absorptions that are indirectly related to nitrogen content. These absorptions may be related to nitrogen differently in different species or at different sites. – The curvature changes dramatically with leaf water content differences.

29. Methods for Determining Leaf Nitrogen: The Derivative Method (cont’d)

30. As a result of these limitations, the models produced using this technique tend to be site and species dependent. This is problematic, since each time you want to use this technique in a new area, ground samples must be collected and chemically analyzed.

31. Methods for Determining Leaf Nitrogen: Continuum Removal In 1999, Ray Kokaly and Roger Clark presented a new technique that they had developed that solved the problems of species and site dependence. Their new approach was based on a technique that is widely used in geological spectroscopy, known as continuum removal.

32. Methods for Determining Leaf Nitrogen: Continuum Removal (cont’d) Continuum removal is implemented by drawing a straight line on a spectral reflectance curve between the beginning and end of an absorption you are interested in. Next, the reflectance at each wavelength of interest in the spectral reflectance curve is divided by the value of the line at that wavelength.

33. Methods for Determining Leaf Nitrogen: Continuum Removal (cont’d)

34. The values are normalized by either the area under the line or the maximum distance between the line and the curve. The main purpose of performing continuum removal is that it helps reduce the affects of absorptions and other spectral features that cause broad trends in the data. In this case, the broad trends that are removed are primarily due to leaf structure and leaf water.

35. Methods for Determining Leaf Nitrogen: Continuum Removal (cont’d) Finally, stepwise multiple linear regression is used to relate the resulting values to foliar nitrogen concentration. The results of this analysis showed that this technique worked very well, with one major exception.

36. Methods for Determining Leaf Nitrogen: Continuum Removal (cont’d) The Achilles heel of this approach is that it is unable to cope with green leaves, since the algorithm is unable to handle large amounts of leaf water. This is because the effect of leaf water on a leaf spectrum is not a linear function of wavelength.

37. Methods for Determining Leaf Nitrogen:The Hybrid Method After Kokaly and Clark’s approach was made public, several researchers attempted to modify the approach to work with green leaves. The obvious solution (and the one suggested by Kokaly and Clark) was to: – Estimate the leaf water concentration – Use this information to create the absorption spectrum of the leaf water present in the leaves being measured. – Subtracted out the leaf water absorption spectrum. This sounds easy, but in reality it is very hard to do well. To the best of my knowledge, no one has gotten this approach to work.

38. Methods for Determining Leaf Nitrogen:The Hybrid Method (cont’d) In the summer of 2001 I came up with a different approach to solving the leaf water problem. It is based on combining the traditional derivative method with Kokaly and Clark’s method.

39. Methods for Determining Leaf Nitrogen:The Hybrid Method (cont’d) The first step in implementing my approach is to reduce the spectral resolution to 10nm by averaging channels. This was done to reduce noise. Next, the values are converted from reflectance to absorption using Beer’s law.

40. Methods for Determining Leaf Nitrogen:The Hybrid Method (cont’d) Once the absorption values have been calculated, a transform is applied to all the channels in the area of the protein absorption features discussed earlier in this presentation. The transform simply involves subtracting the absorption in the middle channel from the average of its two immediate neighbors.

41. Methods for Determining Leaf Nitrogen:The Hybrid Method (cont’d) This is similar to applying a series of continuum removals to the data:

42. Finally, stepwise multiple linear regression is used to relate the transformed values to foliar nitrogen concentration. The idea behind this method is that, like continuum removal, the transformation removes the effects of broad absorption features. However, like the derivative method, it is very localized and emphasizes curvature. My hope was that this method would allow changes in curvature directly related to nitrogen concentration to isolated. Methods for Determining Leaf Nitrogen:The Hybrid Method (cont’d)

43. Testing the Hybrid Method I tested the hybrid method by using data collected for the Accelerated Canopy Chemistry Program (ACCP).

44. Testing the Hybrid Method (cont’d) The dried samples collected from eastern forests were used as training data. The fresh samples of western tree species were used for testing.

45. Testing the Hybrid Method (cont’d)

46. Fresh Dried

47. Testing the Hybrid Method (cont’d)

48. Noise The one major drawback with my method is that the technique is very sensitive to noise. This not an intractable problem at all, but some research will be required to find the best means of collecting low-noise spectra.

49. Conclusions and Future Research Reflectance spectroscopy presents a means of accurately measuring foliar nitrogen concentrations in the field. Unlike other methodologies, my hybrid technique is site and species independent and works with green leaves.

50. Conclusions and Future Research (cont’d) In the future I am planning to use this technique together with spatial statistics to create a nitrogen concentration map for a commercial pine plantation. I also plan to use this technique for additional chemical measurements, including leaf lignin and leaf energy content. Additional research could focus on algorithm improvement and data collection issues.