Fluorescence detection assembly for determination of significant vegetation parameters

• US 6,563,122 B1
• Filed: 04/30/2001
• Issued: 05/13/2003
• Est. Priority Date: 10/28/1998
• Status: Expired due to Fees

• Current Assignee: Deutsches Zentrum Fã¼R Luft- Und Raumfahrt E.V.
• Original Assignee: Deutsches Zentrum Fã¼R Luft- Und Raumfahrt E.V.
• Inventors: Lüdeker, Wilhelm, Günther, Kurt, Dahn, Hans-Günter
• Primary Examiner(s): Hannaher, Constantine
• Assistant Examiner(s): MORAN, TIMOTHY J
• Application Number:
  US09/830,697
• Time in Patent Office: 743 Days
• Field of Search: 250/458.1, 250/459.1, 250/461.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/635 Photosynthetic material analysis, e.g. chrorophyll
• CPC Class Codes: G01N 2021/8466 Investigation of vegetal material, e.g. leaves, plants, fruits
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/0612 Laser diodes

Abstract

A basic assembly configuration for determination of relevant vegetation parameters includes a high repetitive pulsed excitation laser (1) for the stimulation of chlorophyll fluorescence, a fluorescence detector (3) including an imaging and separation optic (4, 5), a trigger and delay electronic (6) to adjust the laser and appropriate detector timing and a detector electronic (7), consisting of a signal recording and processing module. The assembly can be used for determination of relevant vegetation parameters for agricultural, horticultural and greenhouse application.

Claims

1. Fluorescence detection assembly for determination of relevant vegetation parameters comprising an excitation source (1) consisting in a low power laser device with an excitation wavelength in the red spectral region, a beam forming optical device (2), a dichroic beam splitter (5), a basic fluorescence detector system (3) including an entrance optical device (4) receiving fluorescence emission via said dichroic beam splitter (5) and an interference filter (10) blocking out the elastic back scatter signal, an electronic detection device (7) for detecting a fluorescence signal, andy an electronic trigger and timing device (6), characterized in that said low power laser device provided in the excitation source (1) is a high repetitive pulsed laser device with several nanosecond pulse length and a preferred excitation wavelength in the red spectral region of preferable 670 nm, in that said dichroic beam splitter (5) couples the formed excitation beam co-axially to the optical axis (9) of a receiver optic and directing this formed beam without optical waveguiding to a vegetation target subject to be investigated, in that said basic fluorescence detector system (3) forms an image of the excitation spot at the sensitive detector area, in that said electronic detection device operates at the doubled pulse repetition rate of said excitation laser source (1) and samples the active fluorescence signal synchronously with the laser emission on the one hand and the passive background signal with a fixed delay in the microsecond range before or after the active signal on the other hand, recording those signals by means of a fast sample and hold circuit coupled to an analog to digital converter which enables a digital signal processing, in that said electronic detection device (7) further comprises means for determining the pure fluorescence signal by subtracting the background signal from the active fluorescence signal electronically or in a post processing procedure, and in that said electronic trigger and timing device (6) synchronizes the laser pulses with the sample intervals of said electronic detection device (7).

2. Fluorescence detection assembly according to claim 1 wherein said laser device (1) is a laser diode operating in the red spectral region.

3. Fluorescence detection assembly according to claim 1 or claim 2 wherein the power of said laser device (1) is chosen >

0.5 W peak power.

4. Fluorescence detection assembly according to claims 1 or 2 wherein a second laser is added for monitoring blue and/or green fluorescence, the excitation wavelength of said second laser being fixed in the range from 350 to 400 nm, with similar power and timing specifications as the red laser excitation source.

5. Flourescene detection assembly according to claim 3 wherein a second laser is added for monitoring blue and/or green flourescene, the excitation wavelength of said second laser being fixed in the range from 350 to 400 nm, with similar power and timing specifications as the red laser excitation source.

6. Fluorescence detection assembly according to claim 1 wherein said beam forming device (2) comprises an astigmatism correction lens and a beam expander/reducer to a point spot.

7. Fluorescence detection assembly according to claim 1 wherein said fluorescence detector (3) is a photo multiplier tube operating in continuous mode.

8. Fluorescence detection assembly according to claim 1 wherein said fluorescence detector (3) is an avalanche diode.

9. Fluorescence detection assembly according to claim 1 wherein said fluorescence detector (3) is a standard photo diode.

10. Fluorescence detection assembly according to claim 1 wherein said entrance optical device (4) comprises in its aperture a spheric lens, which forms an image of the excitation spot at the masked sensitive area of said fluorescence detector (3).

11. Fluorescence detection assembly according to claim 1 wherein the center wavelength and bandwidth of said interference filter (10) is chosen appropriately in the range from 680-740 nm, the blocking quality beside the transmission being more than 10⁻

3.

12. Fluorescence detection assembly according to claim 1 wherein said sample and hold circuit has an analog bandwidth chosen appropriately between 40 and 200 MHz.

13. Fluorescence detection assembly according to claim 1 wherein said basic fluorescence detector system (3) is supplemented by additional detector elements (13), and additional dichroic beam splitters (11) are provided to the optical axis (12) at the detection side to form specific fluorescence detection bands, the number, center wavelength and bandwidth thereof depending on the scope of investigation.

14. Fluorescence detection assembly according to claim 13 wherein said basic fluorescence detector system comprises two detection devices having first and second detection channels located at 680-690±

5 nm and 720-740±

5 or 10 nm, respectively.

15. A method for determination of chlorophyll concentrations with the fluorescence detection assembly according to claim 14, comprising dividing background corrected signals, determining changes of the relative chlorophyll content per leaf area, and determining the absolute chlorophyll concentrations by multiplying the ratio of fluorescence emission in the first channel to fluorescence emission in the second channel with a predetermined calibration factor.

16. A method comprising determining the chlorophyll status of plants and thus describing the growth state or long term stress conditions of plants, with the fluorescence detection assembly according to claim 14 together with greenhouse robots.

17. A method comprising determining the state of maturity of any chlorophyll containing material when specific fruits lose their typical green color, with the fluorescence detection assembly according to claim 14.

18. A method comprising observing changes in the chlorophyll concentration any chlorophyll containing material whereby decay monitoring of fresh fruits, if they contain chlorophyll in their skin, and vegetation is practicable, with the fluorescence detection assembly according to claim 14.

19. Fluorescence detection assembly according to claim 14 wherein said basic fluorescence detector system comprises a detector head located at a movable “

robot”

arm (X-Y components) which is mounted at a moving platform which provides a third movement component (Z-component).

20. A method comprising controlling site specific fertilization with the fluorescence detection assembly according to claim 19, said step of controlling being based on the fact that chlorophyll concentration of leaves is dependent and thus correlated on the nitrogen and sulfur supply of the whole plant so that fertilizer deficiencies are visible by characteristic reduction and distributions of the chlorophyll concentration which effect is accompanied by reduced growth speed and characteristic changes in the plant density.

21. A method carried out with the fluorescence detection assembly according to claim 19, comprising regulating the vertical position of the “

robot”

arm by a light bar which determines an actual upper canopy level and wherein the detector head is adjusted relative to the upper canopy level, or moves between soil and the upper canopy level.

22. A method comprising monitoring the three dimensional profile of the relevant vegetation parameters with the fluorescence detection assembly according to claim 19, wherein said monitoring step is carried out to take into account the horizontal movement of said moving platform.

23. Fluorescence detection assembly according to claim 19, further comprising an additional excitation source exciting efficiently the blue fluorescence and performing an inter-calibration of the two excitation sources to normalize the fluorescence intensities to the excitation pulse power, said assembly further comprising a detector module with a center wavelength in the range between 430 and 460 nm (Δ

λ

=10-50 nm).

24. Fluorescence detection assembly according to claim 23 wherein said additional excitation source is a triplet Nd:

YAG laser 355 nm.

25. Fluorescence detection assembly according to claim 23 comprising additional detector modules monitoring the green fluorescence (500-550 nm;

Δ

λ

=10-50 nm) to improve the knowledge about the spectral emission feature of the occurring plant types.

26. Fluorescence detection assembly according to claim 23, further comprising a PAR sensor to monitor the illumination environment and thus to interpret band ratios with regard to environmental light conditions.

27. A method for differentiation between monocotyledon and dicotyledon plants with the fluorescence detection assembly according to claim 23, comprising distinguishing the monocotyledon (MC) and dicotyledon (DC) plant types by their characteristic feature of the fluorescence emission spectra from 400 to 750 nm, said step distinguishing comprising measuring the blue fluorescence relative to the red chlorophyll emission.

28. The method of claim 27, further comprising providing a predetermined ratio threshold defining whether a signal belongs to a MC or DC plant.

29. The method of claim 27 comprising providing a classification algorithm, in the case of programmable post processing, defining whether a signal belongs to a MC or DC plant.

30. A method for performing site specific herbicide treatment in terms of weed position and type, comprising detecting relevant vegetation parameters with the fluorescence detection assembly according to claim 23.

31. A method for detection of various fungal infections, comprising:

detecting relevant vegetation parameters with the fluorescence detection assembly according to claim 23 extended by the second detection band in the red fluorescence region; and

using one detector package to monitor the elastic back scatter signal.

32. Method according to claim 31, further comprising recognizing effects on the photosynthetic system by changes in the fluorescence intensity.

33. Method according to claim 31, further comprising further comprising determining changes in leaf morphological structure, total cell destruction or variations in the composition of plant pigment constituents by determining the channel ratio of the red detection bands, and provides an additional quantitative identification criterion based on inhomogeneous infections and their characteristic distribution over total leaf area or canopy.

34. Method according to claim 31 further comprising recognizing changes in the composition of blue and/or green fluorescence emitting pigments as a result of infection by the detection channels in this range, for discovering some fungi that are characteristic sources of blue and/or green fluorescence.

35. Method according to claim 31, further comprising recognizing leaf surfaces that are covered with an additional tissue layer causing a significantly increased surface reflectance, by observing the elastic back scatter signal at the red as well as at the UV excitation wavelengths.

36. Method according to claim 31 applied to site specific fungicide treatment based on fungi position and type.

37. A method comprising monitoring the photosynthetic activity of vegetation by time series with the fluorescence detection assembly according to claim 14, wherein the investigated target is variable.

38. Fluorescence detection assembly according to claim 1 further comprising two additional detectors recording the elastic back scatter signal at the emission wavelengths of the laser sources.

39. Fluorescence detection assembly according to claim 1 comprising an additional photodiode, installed in the optical path at the excitation system monitoring the laser pulse energy.

40. Fluorescence detection assembly according to claim 1 comprising a PAR(photosynthetic active radiation)-sensor located above the measurement position monitoring the environmental light conditions.

41. Fluorescence detection assembly according to claim 1 comprising a vertically movable, horizontally oriented light bar determining the canopy top level and thus plant height, the vertical position of the basic fluorescence detector system being coupled with said light bar and being fixed or moving relative to it.

42. Fluorescence detection assembly according to claim 41 comprising hydraulic, pneumatic or mechanical elements realizing the movement of said light bar as well as said basic fluorescence detector system.

43. Flourescene detection assembly according to claim 1 wherein said beam forming device (2) comprises a cylinder lens and a beam expander/reducer to a point spot.

44. Fluorescence detection assembly according to claim 1, further comprising a detector head mounted to a movable robot arm, and a moving carrier platform to which said robot arm is fixed, and wherein said laser device is an excitation laser diode operating at 670 nm, said basic fluorescence detector system comprises a detector module, and said interference filter transmits at a wavelength in the range from 680 to 740 nm with a spectral bandwidth between 5 and 25 nm.

45. A method for detection of chlorophyll containing plants or plant organs (plant recognition), i.e. green vegetation, with the fluorescence detection assembly according to claim 44, comprising using a suitable threshold to recognize vegetation, such that the contrast between vegetated and non vegetated targets is sufficiently high that a recognition is done without any further signal processing.

46. A method for determination of relevant vegetation parameters with the fluorescence detection assembly according to claim 44, comprising defining a plant position relative to the position of the detector head and determining the distribution of plant material by assuming a controlled two or three dimensional movement of the carrier platform.

47. A steering system for greenhouse or horticulture robots comprising the fluorescence detection assembly according to claim 44.

48. A method for detecting and destroying plants comprising:

detecting plants with the fluorescence detection assembly according to claim 44, and destroying plants that are detected.

49. A method comprising monitoring the photosynthetic activity of vegetation by time series with the fluorescence detection assembly according to claim 44, wherein the investigated target is fixed and thus the variation is only time dependent.

50. Method according to claim 49 or claim 37, further comprising determining the environmental light condition utilizing a PAR sensor.

51. Method according to claim 49 or claim 37 wherein digitized fluorescence values are interpreted with respect to the existing illumination situation and history while post processing.

52. Method according to claim 49 for scientific investigation of long term processes, comprising investigating the interaction of plants and variable environmental conditions.

53. Method according to claim 49 for scientific investigation of the Kautzky kinetic and all related parameters which can only be measured at a dark adapted plant, at a fixed position.

54. Method according to claim 53, comprising utilizing the excitation source as a source of actinic light and increasing repetition rate of pulses from the excitaiton source to induce an actinic reaction.

55. Method according to claim 53, comprising utilizing a triggering extra white light source, which illuminates the vegetation at a determined time, as a source of actinic light.

56. Method according to claim 49 for scientific investigation of the Genty-Parameter, comprising measuring steady state fluorescence as well as steady state fluorescence superimposed on a saturating light pulse.

57. Method according to claim 56, comprising exciting the steady state fluorescence (Fs) remotely by a laser diode while using an additional white lamp or the sun to act as actinic light, and generating the saturating light pulse (Fm′

) by an additional light source such for monitoring Fs and Fm′ and

the intensity of the saturating light based on detection of a passive background signal.