A passive two-band sensor of sunlight-excited plant ﬂuorescence Paul L. Kebabian, Arnold F. Theisen,

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A passive two-band sensor of sunlight-excited plant fluorescence

Paul L. Kebabian, Arnold F. Theisen,^a)Spiros Kallelis, and Andrew Freedman^b)

Center for Materials Technology, Aerodyne Research, Inc., Billerica, Massachusetts 01821-3976 共Received 20 April 1999; accepted for publication 30 July 1999兲

We have designed and built a passive remote sensor of sunlight-excited chlorophyll fluorescence 共U.S. Patent No. 5,567,947, Oct. 22, 1996兲 which provides for the real-time, in situ sensing of photosynthetic activity in plants. This sensor, which operates as a Fraunhofer line discriminator, detects light at the cores of the lines comprising the atmospheric oxygen A and B bands, centered at 762 and 688 nm, respectively. These bands also correspond to wavelengths in the far-red and red chlorophyll fluorescence bands. The sensor is based on an induced fluorescence approach; as light collected from fluorescing plants is passed through a low-pressure cell containing oxygen, the oxygen absorbs the energy and subsequently reemits photons which are then detected by a photomultiplier tube. Since the oxygen in the cell absorbs light at the same wavelengths that have been strongly absorbed by the oxygen in the atmosphere, the response to incident sunlight is minimal. This mode of measurement is limited to target plants sufficiently close in range that the plants’ fluorescence is not itself appreciably absorbed by atmospheric oxygen 共⬃200 m兲. In vivo measurements of fluorescence in the 760 and 690 nm bands of vegetation in full sunlight are also presented. Measurements of plant fluorescence at the single-plant canopy level were obtained from greenhouse-grown bean plants subjected to a range of nitrogen treatments. The ratio of the fluorescence obtained from the two measurement bands showed statistically significant variation with respect to nitrogen treatments. © 1999 American Institute of Physics.

关S0034-6748共99兲01511-7兴

I. INTRODUCTION

The intensity and spectral band shape of chlorophyll fluorescence in green plants has been linked to the physiological status of the plants and thus provides a good indicator of general plant health.¹Chlorophyll fluorescence am- plitudes have been shown to be affected by a number of factors including nutrient and water stress, carbon dioxide levels, disease, and pest damage. The relationship between in vivo chlorophyll fluorescence and photosynthetic activity was first described by Kautsky² over 60 years ago and has been extensively used to study photosynthetic activity of both terrestrial plants and marine-based phytoplankton.^3,4 Fluorescence measurements offer great advantages over other ecophysiological methods in that it is nondestructive and applicable to both individual leaves and needles, and canopies.

The efficacy of plant fluorescence sensing as a detector of plant stress relies on the fact that energy from a wide range of the ultraviolet and visible spectrum can be absorbed by chlorophyll and other pigments found in green plants for use by leaves, needles, or even bark. This energy is used for photochemistry共i.e., photosynthesis兲, or dissipated through a number of other mechanisms 共including fluorescence兲, of which the major one is the production of heat共with the par- ticipation of xanthophyll molecules兲.⁵ At moderately high

light levels 共800 ␮^{mol m}^⫺²^s^⫺¹兲, 10%–50% of the incident light energy is used for photosynthesis, and in full sunlight 共2000 ␮^{mol m}^⫺²^s^⫺¹兲, the percentage is even lower. When plants are exposed to stress 共excess heat or cold, nutrient deficiency, herbicides, etc.兲, their rate of photosynthesis drops and the fraction of absorbed light energy shunted to the other dissipative mechanisms increases. Thus, the premise behind measuring plant fluorescence is that fluorescence intensity is related 共albeit in a complicated way兲 to photosynthetic activity, thereby providing an indicator of physiological activity.

In practice, it has been determined that the measurement of the ratio of fluorescence intensity at two wavelengths共in- stead of the more difficult task of measuring absolute inten- sities兲 provides a useful measure of plant stress. Incubation of a leaf with the herbicide diuron 共DCMU兲, for example, increases both red and far-red 共R/FR兲 emission intensities 共peaking at 690 and 735 nm, respectively兲, although red emission is increased to a greater degree than is far-red emission. Diuron acts to disrupt the electron-transport pathway in the photosynthetic mechanism, minimizing photosynthesis and maximizing the dissipative processes including fluorescence.⁶ The effectiveness of the fluorescence ratio of 690–735 nm 共R/FR兲as a measure of plant stress in relation to electron transport, is attributed to the fact that the far-red emission is less responsive than red emission to changes in photosynthesis rates at normal physiological temperatures.⁷ It is generally accepted that the fluorescence peak in the red portion of the spectrum near 685 nm is principally due to photosystem II 共PSII兲emission; the fluorescence peak near

a兲Present address: Virginia Institute of Marine Science, School of Marine Science, RMAP, CERSP, Gloucester Point, VA 23062-1346.

b兲Corresponding author; electronic mail: af@aerodyne.com

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735 nm is thought to comprise a combination of emissions from both photosystems I and II共PSI and PSII兲, although the relative contribution is a matter of some disagreement.^8–13

The relationship between the ratio of fluorescence emission at 690 nm to that at 735 nm and plant chlorophyll content has also been established for several plants.¹⁴ For Nor- way spruce, this red/far-red ratio varies inversely with chlorophyll content which increases up to a point as a plant matures, and decreases under conditions of stress, such as mineral or water shortage or excess, as well as high light or heat levels. The increase in the 690 nm/735 nm ratio with loss of chlorophyll is ascribed to a decrease in self- absorption by chlorophyll in the red spectral region.³

The measurement of chlorophyll fluorescence emission has generally been performed with active stimulation 共such as flash lamps, light-emitting diodes, and pulsed lasers兲using laboratory spectrophotometers, induction-kinetic instruments, and spectral radiometers and imaging systems.^15–18 However, the passive measurement of in situ solar- stimulated plant fluorescence has proved to be a more difficult task because the intensity of plant fluorescence, under the best of circumstances, is 2–3 orders of magnitude less than that of the solar flux. A limited number of measurements of this type have been conducted with field and laboratory instruments using a combination of atmospheric and solar absorption features based on the Fraunhofer line-depth method.^19–22 However, widespread utilization of this technique has been hindered by the lack of instrumentation suit- able for use outside of the laboratory environment.

In response to this need, we have designed and built a robust sensor of sunlight-excited chlorophyll fluorescence that provides for the real-time, in situ, and remote共⬃200 m range兲 sensing of photosynthetic activity in plants.²³ This sensor operates as a Fraunhofer line discriminator, detecting light at the cores of the lines comprising the atmospheric oxygen A and B bands, centered at 762 and 688 nm, respectively.²⁴ These bands also correspond to wavelengths in the far-red and red chlorophyll fluorescence bands. The sensor operates, without use of an interferometer or spectrometer, as an induced fluorescence detector—that is, as light collected from the fluorescing plants is passed through a cell containing oxygen at low pressure, the oxygen absorbs some of the incident energy and subsequently reemits photons which are detected by a photomultiplier tube. This induced fluorescence signal is directly proportional to the absolute intensity of the plant fluorescence in the narrow oxygen absorption bands. Furthermore, since the oxygen in the cell absorbs light at exactly the wavelengths that are strongly absorbed by oxygen in the atmosphere, the response to residual incident sunlight is minimal. This instrument provides not only the ratio of fluorescence intensities in the two spectral bands, but also absolute fluorescence intensities.

II. INSTRUMENT DESCRIPTION A. Operating principles

In the classical Fraunhofer line discriminator共FLD兲, the intensity of light from a target is observed at a wavelength where there is an absorption feature in the solar spectrum

共i.e., a Fraunhofer line兲using a high-resolution spectrometer and compared to an adjacent spectral region outside the absorption line.^25–28The apparent depth of the absorption line will be reduced by any fluorescence from the target, which is assumed to be continuous and free of narrow absorption or emission lines. Prior use of this technique for the measurement of plant fluorescence has been limited to utilizing the hydrogen-␣ ^line 共656.3 nm兲 at which wavelength the fluorescence spectrum of chlorophyll is weak. Moreover, the H-␣line still has⬃10% of the off-line intensity, even at its center;²⁹ thus, H-␣-based FLDs might have to measure the chlorophyll fluorescence as the small difference between two large quantities. In contrast, the FLD reported in this work uses instead the fully opaque absorption lines of atmospheric oxygen in its A band共centered at 762 nm兲and B band共cen- tered at 688 nm兲. The fluorescence emission, at the cores of the oxygen absorption lines, is measured against a negligible background; at most, the residual contribution from scattered sunlight is a small correction to the measured fluorescence of the target.

The relationships among the spectra involved in the operation of the plant fluorescence sensor are illustrated in Fig.

1. The top panel shows a typical spectrum of chlorophyll fluorescence which is the superposition of several bands corresponding to different chemical environments of the chlorophyll molecules, including bands peaking near 680 and 740 nm.⁵ The observed chlorophyll fluorescence spectrum includes the effect of reabsorption of the fluorescence in the leaf; below approximately 700 nm, chlorophyll absorbs strongly, while at longer wavelengths, it absorbs weakly.

Also shown in the top panel are the approximate locations of the atmospheric oxygen bands, the A band near 762 nm and the B band near 688 nm. 共The actual A band absorption is approximately 12 times that of the B band.兲Note that both these bands are in regions of strong chlorophyll fluorescence and are spaced on either side of the absorption edge. The middle panel shows the transmission spectrum of the atmo- sphere in the A-band region, for sunlight incident at a zenith angle z⫽48° 关air mass ⬃sec 共z兲兴. This spectrum was com- puted using the tabulated spectroscopic parameters of oxygen,^30,31and agrees closely with the measured spectrum of Ref. 29.

The instrument described here also differs from past at- tempts to use a FLD to measure plant fluorescence in that no form of spectral light dispersion is attempted. Instead, an induced fluorescence technique is utilized. To observe the light at the cores of these lines alone, our FLD uses a cell containing low-pressure oxygen as a molecular line filter; the oxygen in the cell absorbs light from the target and subsequently reemits part of the absorbed light as fluorescence.

This technique relies on the fact that the A and B bands are vibrational–rotational bands of an electronic transition of the oxygen molecule (X³⌺g⫺→b¹⌺g⫹, v⬘^⫽0,1). After absorb- ing a photon in the A 共or B兲band, the oxygen molecule can reemit a photon in the A band, which can then be detected by a photosensitive device. The solar blind nature of this detection scheme is demonstrated in the lower panel in Fig. 1, which shows an enlarged view of part of the atmospheric transmission spectrum, superimposed on that which is the

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relative absorption coefficient of low-pressure oxygen gas, such as that present in the absorption cell. Note that the atmospheric lines show negligible transmission at line center, and that the low-pressure lines show minimal absorption in the region where there is appreciable atmospheric trans- mission. The spectrum in the B-band region has a similar appearance, but the band strength is weaker by a factor of approximately 12.²⁴

B. Equipment

The plant fluorescence sensor共shown in schematic form in Fig. 2兲functions as a highly efficient radiometer comprising a 6 in. i.d. high-reflectivity integrating sphere 共Lab- sphere, North Sutton, NH兲, radiometrically calibrated silicon photodiode, a cooled red-sensitive photomultiplier tube, a three-position, manually or computer-controlled interference filter holder which acts to spectrally limit the observed radiation, and two precision mechanical choppers. The 1 in. o.d.

aperture to the integrating sphere is compatible with up to

f /2 light collection optics; quartz and acrylic light pipes are also used to improve light-coupling efficiencies among the various components of the sensor. The photodiode measures the total in-band共oxygen A or B band兲radiation received by the integrating sphere depending on which interference filter covers the aperture of the sphere. The third position in the filter holder is blanked off, allowing for the measurement of any electronic offsets or light leakage into the sensor.

In order to measure the plant fluorescence, a 6 in. o.d.

quartz bulb, filled with a low-pressure共⬃100 Torr兲mixture of oxygen共5%兲and argon, is placed within the cavity of the integrating sphere. Given that the efficacy of oxygen as a molecular line filter is limited by its small absorption cross section and long emission lifetime共⬃12 s兲,^24,30,31great care must be taken to maximize the induced fluorescence signal.

Collisional deexcitation by background gas and the cell walls is minimized by using a large cell, a low total pressure, and an inert buffer gas 共argon, which has an extremely low quenching rate constant兲.³² In order to eliminate avoidable collisional quenching, maintenance of rigorous gas purity is required; water vapor concentrations above the low parts per billion range must be avoided. All gas filling of the bulb is executed using a 0.25 in. o.d. sidearm tube that contains a quartz-to-annealed-copper transition. The bulb is then sealed by ‘‘pinching off’’ the annealed copper tube using a tool that is specially designed for this purpose. Using this technique, no degradation of the gas mixture has been observed. The cell response, as determined by measuring the apparent emission lifetime, has remained constant over the past 12 months, indicating that no atmospheric or virtual leakage has taken place. This sealing technique avoids the possible contamina- tion issues raised by the more usual technique of using a torch to seal off a constricted quartz fill tube. It also maxi- mizes the pumping speed through the fill port.

Two high-discrimination light choppers are placed at the entrance and exit ports of the integrating sphere. During operation, 760 or 690 nm light emanating from both plant fluorescence and ambient sunlight 共selected using 10 nm bandwidth interference filters兲, irradiates the oxygen in the quartz cell while the entrance chopper is open; during this stage the exit chopper is closed. After the entrance chopper blocks the

FIG. 1. Relationship between chlorophyll fluorescence and the atmospheric oxygen bands. The top panel shows the chlorophyll fluorescence spectrum with the location of the oxygen A and B bands marked. The middle panel presents the transmission spectrum of the atmosphere in the region of the oxygen bands calculated at air mass⫽1.5. The bottom panel shows the shapes of two individual oxygen lines at a total pressure of 0.1 atm compared to that of the atmospheric transmission.

FIG. 2. Schematic diagram of the plant fluorescence sensor.

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light source, the exit chopper opens and the emitted photons 共primarily generated from plant fluorescence兲are detected by a cooled photomultiplier tube 共PMT兲. The resulting fluorescence decay curve is generated by collecting the photo- current-generated pulses. During the excitation phase, a silicon diode monitors the incident light intensity in the integrating sphere. A current-to-frequency converter changes this signal to a pulse train, which is acquired by a counter.

The next three subsections present more complete de- scriptions of the high light-rejection choppers, the data acquisition system, and the software required to collect the data.

1. High light-rejection choppers

The relevant signal levels in this instrument can be as low as tens of detected photoelectron pulses per second.

Thus, it is critical that the input chopper fully blocks the incident light when in the closed phase. 共Since the function of the output chopper is solely to protect the PMT while the input chopper is open, its performance is less critical.兲More- over, the choppers must handle a relatively large diameter beam 共1.0 in.兲, which subtends a large solid angle, since the output of the integrating sphere is essentially isotropic over an entire hemisphere. To achieve these goals, the chopper design incorporates the following optical/mechanical design features:

共a兲 A fully enclosed chopper disk of 8 in. diam, with a single window comprising half of the circumference. The objective of this is to maximize the window opening relative to the beam size; since this design is not balanced geometri- cally, material is removed from the closed half of the blade, and final balance is achieved with small added weights.

共b兲 A labyrinth seal around the entire perimeter of the chopper disk. This acts to suppress light leakage through the short path around the outer edge of the disk, by forcing scattered light to make many reflections before escaping from the seal. Figure 3 shows the geometry of this seal, which is built with clearances of approximately 0.03 in. between the cylinders of the blade’s labyrinth and those of the housing.

共c兲 Light is brought to and from the chopper blade using 1 in.-diam light guides 共both quartz and acrylic plastic have been used for this兲. The gap to the light pipe is approximately 0.06 in. on each side of the chopper disk. This mini- mizes the light that misses the output coupling aperture of the chopper.

This chopper design provides a usable transmission window 共as a fraction of a revolution兲 of 0.4, i.e., 80% effi-

ciency. In the closed state, the residual transmission at the ends of a window of similar duration is ⬍10^⫺⁹, falling to

⬍2⫻10^⫺¹⁰at the midpoint of the window.

The choppers are driven by brushless dc motors originally manufactured for use in standard 3.5 in. floppy drives for data storage. As originally made, the speed is controlled to 10 revolutions/s by phase locking to a locally generated 1.024 MHz frequency. This is further divided to 400 Hz, and the motor speed controller locks the signal from the serpen- tine speed sensor winding of the motor to that lower reference frequency. The motors were modified by adding a magnetic pickoff to provide a reference pulse once per revolution, coinciding with the chopper being exactly half open. Also, the local reference frequency was replaced by an externally generated 1.0 MHz signal 共and thus, the exact chopper speed is 9.77 revolutions per second兲. To phase lock the two choppers, the reference pulses are compared digi- tally, and for whichever chopper is leading, the reference frequency signal is interrupted for 100␮s once per revolution. When the two reference pulses coincide within a 500␮^s window, both motors receive an uninterrupted 1 MHz reference frequency. This method of locking the two choppers was found to be more stable and less subject to upset from noise spikes than a phase lock based on a standard 4046 phase lock chip.

2. Signal detection and data acquisition

The PMT used in this instrument 共type 9828, Electron Tubes, Inc., Rockaway, NJ兲 is enclosed in a thermoelectri- cally cooled housing共Products for Research, Danvers, MA兲, which maintains the tube at 40° below ambient. Since the cathode of the PMT is located several inches behind the exit plane of the chopper, light is conducted to it via an acrylic light pipe. This passes through a dual O-ring seal that re- places the window originally supplied with the housing. The light pipe is coupled to the PMT cathode using a silicone rubber optical coupling disk共Bicron, Newbury, OH兲. Acrylic plastic is a very poor thermal conductor; this arrangement is found to not measurably increase either the dark rate of the PMT or the indicated temperature of the cooled housing. The power for the cooled housing共8 A at 3.5 V兲is obtained from a switching power supply共designed for use with 3.3 V digital circuitry兲, which also provides the 12 and 5 V supplies for other circuitry. The cooled housing has extensive electro- magnetic shielding, and no problem has been encountered with switching spikes from the power supply. The high- voltage supply to the PMT is enabled only when the two choppers are locked. As further protection, the current output of the high-voltage supply is monitored and the high voltage is shut down if a threshold, approximately 20␮^{A over the} normal divider chain current, is exceeded.

The signal from the PMT anode is sent to a charge amplifier/discriminator共A101, Amptek, Bedford, MA兲. This discriminator is used at its preset threshold setting of 10⁶ electrons. In order to optimize the final signal-to-noise ratio of the measurement, the ratio of the PMT signal counting rate to the square root of the dark counting rate was determined as a function of the high voltage supplied to the PMT.

FIG. 3. Schematic detail of labyrinth seal of chopper blade. Both blade and housing are spray painted flat black over a black anodized aluminum base.

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The optimum voltage was determined to be approximately 100 V above the 200 A/lm value specified in the test data supplied with each PMT.

When the input chopper is open, the illumination in the integrating sphere is monitored with a silicon photodiode.

The current from this diode is converted to a voltage and then transformed to a variable-frequency pulse train using an LM331 voltage-to-frequency converter 共VFC兲 chip. Thus, both the illumination and PMT signals consist of pulse trains that are acquired by digital counters. The counters used here consist of two 82C54 counter chips, each of which provides three programmable 16-bit counters. These are located on a board 共PPIO-CTR06, Computer Boards, Inc., Middleboro, MA兲 that interfaces them to the parallel port of a computer used for data acquisition. This board was modified to provide an interrupt to the host computer.

In operation, the synchronization pulse from the choppers resets a first counter that begins counting down, and generates an interrupt to the host PC every 6 ms thereafter.

On each interrupt, the PC reads the contents of the PMT pulse counter, and subtracts the previous reading to arrive at the number of detected photons during that time bin. In each of 17 time bins, the total number of pulses is accumulated.

Since the next synch pulse from the chopper resets the bin timer counter before the 18th interrupt pulse is generated, this process repeats on each revolution of the chopper. After 17 s observation, i.e., 166 revolutions of the chopper, the total observing time of each bin is 1 s, a convenient 共but arbitrary兲value that makes the total accumulated pulses correspond to the average rate共pulses per second兲. At that time, the contents of the VFC counter also are read out.

The interference filter 共A band, B band, or dark兲 is selected using a filter changer 共Optec, Inc., Lowell, MI兲. The controller for this filter changer responds to a width- modulated pulse train that is generated by other counters on the counter board. After selecting each filter, the pulse width is reset to an inactive value, so that the manual control inputs remain operable, as well as software control via the pulse train.

The contents of the first and last PMT counter bins are discarded, as they are subject to light leakage at the start of the input-chopper-closed phase. Thus, bins 2–8 contain the pulse counts from which the fluorescence of the oxygen con- tained in the integrating sphere is measured. Moreover, the time resolution of these 6 ms time bins allows one to determine the fluorescence decay time constant of the oxygen cell, a key indicator of the health of the instrument. The remaining bins contain dark counts; measurement of the PMT dark rate indicates whether the PMT is adequately cooled, not subject to electrical breakdown, etc. Further, in the dark filter state, comparison of the counts in bins 2–8 with those in bins 9–16 provides a diagnostic of possible light leakage into the integrating sphere. After each 17 s observation, the data line stored by the PC consists of: a number共1, 2, or 3兲denoting which filter station was in use;

the contents of bins 2–16; and the total VFC pulse count.

3. Software

Several versions of software have been generated, ac- cording to the requirements of the application. The main data

acquisition program is written in Forth language共PC-Forth, Laboratory Microsystems, Inc., Marina Del Rey, CA兲, with interrupt handlers written in x86 assembly language. This program displays each incoming line of data on the screen, accepts keyboard commands of instrument operation, and logs notes and comments entered by the user. The notes are stored with an index that keys each note to the data line at which it was entered. The data, notes, and notes index are saved to disk as a binary file with the required header for reading byMATLAB共Mathworks, Natick, MA兲, in which sub- sequent display and processing routines are written. The main data acquisition program is compact共⬃50 kbytes兲, and can run on virtually any DOS-based PC that is connected to the parallel port connector of the counters.

The data acquisition program has also been adapted to accept commands, and emit each data line, over an RS-232 communications line. In this version, it operates on a compact single-card PC 共KS-9, Kila Systems, Boulder, CO兲that resides inside the fluorescence sensor’s cover. In this version, the logging of comments and data is performed by a PC at the other end of the communications line using a Windows- based shell 共written in Visual Basic兲, which provides a graphical user interface for the user; it also allows the parallel port of that PC to be used as the interface to a digital camera. In field experiments, we have found the digital camera to be a valuable tool to document the scene and illumination conditions of each observation; to minimize parallax, the camera is located directly beneath the input to the filter changer.

C. Data analysis

The sensor contains two measurement elements, the silicon photodiode and the induced fluorescence detector. De- pending on which filter covers the sensor aperture, the silicon photodiode measures the total amount of light in band which enters the sensor 共labeled I for illumination兲. This term is the sum of locally produced fluorescence 共presum- ably from the plant兲and reflected sunlight. The induced fluorescence detector is used to determine what fraction of that light is caused solely by the local fluorescence. Instrument response to purely local fluorescence can be calibrated by using an artificial light source共such as an incandescent lamp兲 which emits a continuous spectrum of light in the regions of the interference filters共i.e., an absence of Fraunhofer lines兲. Thus, the response of the induced fluorescence element for a given in-band photodiode signal can be measured 共labeled C兲. This normalized calibration is performed for both A and B bands since the instrument response depends on the rela- tive absorption strengths of each band, and the bandwidth and transmission of each interference filter. In the absence of any other source of signal, the signal obtained from a plant could simply be compared to the calibration constant C to obtain the total fluorescence. However, a significant base- line signal is observed and must be taken into account.

Two contributions to the base-line fluorescence signal have been established. The first is a small residual response to ambient sunlight. This is a function of the oxygen temperature and pressure and the amount of air mass the sunlight passes through during the measurement. As discussed before,

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this is a small correction. The second and much larger contribution derives from a phosphorescence signal emanating from the polymer material from which the integrating sphere is constructed. The base-line signal 共labeled B兲 can readily be measured by viewing sunlight reflected from a nonfluo- rescent white surface.

Thus, the locally produced plant fluorescence 共PF兲 for each wavelength is simply

PF共␭兲⫽

冋

^C^S^⫺^⫺^B^B

册

^I,

where S is the total raw signal from a plant measurement, C is the calibration共or span兲constant in each band, and B is the base-line 共residual兲 response of the instrument. All these quantities are normalized to the diode signal I. If the diode signal I is radiometrically calibrated, the instrument not only determines the R/FR, but also produces an absolute measurement of the plant fluorescence in units of energy flux per unit wavelength共e.g., W m^⫺²nm^⫺¹in band兲.

Typical fluorescence decay signals are shown in Fig. 4.

One trace shows the time-averaged time decay of the raw signal S in the atmospheric A band共762 nm obtained while viewing grapefruit trees in mid-day sun in March; this signal was collected in approximately 2.5 min of observation time.

The residual response of the instrument B共which includes a contribution from sphere fluorescence兲and the difference of the two signals are also displayed in order to demonstrate the relative intensities of the quantities. The difference signal is integrated to produce the total plant fluorescence. In the A-band region, signal-to-noise ratios 共S/N兲 of 30–50 are readily obtained in full sunlight with integration times of 30 s. However, in the B band, due to the fact that the oxygen absorption is ⬃12 times weaker, S/N’s of only 2–10 are typically obtained. We are currently working to increase the light collection efficiency of the sensor and reduce the inte-

grating sphere fluorescence in order to improve the sensor performance.

III. PLANT FLUORESCENCE MEASUREMENTS

In order to demonstrate the efficacy of the plant fluorescence sensor共PFS兲, a short series of trials were performed in which some common bean plants共Phaseolus vulgaris L.var.

Newport兲were fertilized with various levels of nitrogen fertilizer and observed with the PFS. Nitrogen is an important subject of study with respect to remote-sensing instrumentation because it is a major macronutrient for almost all plant life; in many parts of the world, it is scarce and expensive and in other areas, its overuse has led to problems of con- tamination of ground and surface waters.^33–35 It is both an economic and ecological benefit to be able to detect optimum as well as deficient plant growth conditions. The requirement for the application of supplemental nitrogen fertilizer following a moderate initial application, thus avoiding overuse, can be met by sampling a crop and measuring the nitrogen con- centration in the plants. It is important to note that the preponderance of leaf nitrogen is allotted to thylakoid proteins and the Calvin cycle; nearly half of that is associated with the photosynthetic process, of which chlorophyll is a major component.³⁶In other words, chlorophyll content follows nitrogen availability and uptake.

These experiments involved the growth of six plants using four nitrogen treatments for a total of 24 bean plants, grown in Perlite® in containers modified to retain a nutrient reservoir. The plants were distributed in a random block design across a 4⫻12 ft bench equipped with ebb and flow basins. The plants were fed with a complete hydroponic nutrient solution with the exception that the nitrate and ammo- nium chemical salt quantities were changed to provide for four levels of nitrogen 共30, 60, 90, and 120 ppm兲. The solutions were prepared, individually, in large opaque tanks and delivered to the plants by standard hydroponic drip tubing connected to submersible electric pumps.

A preliminary crop of beans was grown to confirm the suitability of the variety for the experiments that followed.

This crop, designated crop 1, served also to confirm the relationship between nitrogen treatments and the physical changes that the different levels induced. The nonroot portion of this crop was harvested and the dry weights determined. Analysis of variance共ANOVA兲confirms that the dry weights are significantly different by treatment at P⬍0.05. A linear regression analysis yielded an r² of 0.95.

Crop 2, grown and observed over a period from October to November 1997, was transplanted to treatment eight days after seeding. Two sets of measurements with both the PFS and SPAD chlorophyll instruments were made, the first 43 days and the second 49 days after seeding. Chlorophyll content data were collected following each PFS measurement using a Minolta SPAD-502 meter.^37–39 Several chlorophyll measurements were made on each leaf and several leaves per plant were sampled. Fluorescence emission measurements were made in the far-red 共760 nm兲 and red共690 nm兲bands with the plant fluorescence sensor equipped with an f /2 tele- scope rotated to the horizontal position. Plants were placed

FIG. 4. Typical fluorescence decay signals at 760 nm collected from grapefruit trees in mid-day sun in March. Shown are the raw signal, the residual response of the instrument, and the difference signal which is integrated to produce a plant fluorescence signal.

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on a platform⬃2 m from the end of the telescope with the main portion of the biomass centered in the field of view.

Data were collected for each plant for three complete cycles consisting of D共dark, blank plate; no input signal兲, B band, A band, B, B, A, and B. For calibration purposes, the tele- scope was rotated into the vertical position and measurements were taken of both sunlight and a 120 W ac floodlight scattered from a highly reflective diffuse material共a Tyvek®

envelope兲 laid out horizontally. Using sunlight to calibrate that absolute intensity of the diode in this fashion introduces

⫾25% uncertainty due to the estimated uncertainty in the model calculation of the solar flux; relative intensities are measured to better than 10%.

SPAD-based chlorophyll measurements for both days 共shown in the left panel of Fig. 5 as an average of six plants per treatment兲showed a strong positive correlation between leaf chlorophyll levels and nitrogen treatment levels.

ANOVA indicates that the chlorophyll differences as a func- tion of treatment were statistically significant ( p⬍.001) for both measurement days. However, no statistical difference was observed from day to day, an observation which can readily be discerned from Fig. 5. From the dry weight and chlorophyll data, it is clear that the beans grown for these experiments have been affected by the different levels of nitrogen provided in their nutrient solutions and meet the definition of plants under stress.¹ The results are in good agreement with those seen in previous studies.²²

The ratio of red共690 nm兲to far-red共760 nm兲emission 共R/FR兲showed a clear trend as well. ANOVA indicated that R/FR differences with respect to nitrogen treatment levels were also statistically significant ( P⬍0.001). The right panel in Fig. 5 shows R/FR data for both days plotted versus nitrogen treatment levels. It is clear that the R/FR fluorescence ratios show a strong inverse correlation with nitrogen treatment. It is interesting to note that while the chlorophyll contents of the plants were highly correlated with nitrogen treatment levels, the chlorophyll contents of the plants did not vary within the time frame of the two measurements. The chlorophyll contents of the plants were virtually identical on

days 43 and 49 for a given nitrogen treatment共except for the lowest treatment level兲. However, the R/FR measurements show statistically significant differences between days 共ANOVA, p⬍.001). These findings supply preliminary evi- dence that plant fluorescence emission may provide a more timely indicator of plant stress than does chlorophyll content.

Absolute fluorescence intensities 共averaged by treat- ment兲are presented in Fig. 6; the values shown assume that the instrument views an opaque canopy. The statistical error bars for the 690 nm data are far greater than those for the 760 nm data; this is the result of the aforementioned lower signal-to-noise ratios for measurements in the 690 nm band.

True plant-to-plant variations are probably comparable to those observed in the 760 nm band. Since the emission intensities are not corrected for the leaf area in the field of view of the PFS共i.e., the effective opacity is less than 1兲, compar- ing absolute intensities between plants subjected to different treatments is not strictly permitted. However, given that: the preponderance of the plant biomass is in the instrument field of view; the foliage is not particularly dense 共minimizing canopy self-absorption effects; and the amount of biomass is proportional to the nitrogen treatment, it is reasonable to conclude from the data that, on the average, absolute fluorescence intensity increases as a function of the applied stress and the concomitant decrease in the leaf chlorophyll content. Furthermore, the data can be interpreted as showing that the differences in fluorescence ratios on the two days were caused primarily by significant increases in intensities in the 690 nm band. The fact that day-to-day 760 nm band intensities varied only slightly would seem to indicate that fluorescence emission from PSI is not greatly affected by changes in plant physiology caused by stress. However, given that the 690 nm band fluorescence intensities show fairly large increases from one day to the next, one can sur- mise that the observed changes in fluorescence ratios from

FIG. 5. Leaf chlorophyll content 共left panel兲 and fluorescence emission ratios共690 nm/760 nm兲共right panel兲of bean plants as a function of nitrogen treatment. The data points represent the mean values of fluorescence emission ratios 共690 nm/760 nm兲 measured for six plants for each nitrogen treatment level. Observations were made 43 days and 49 days after planting.

Error bars represent one standard deviation of the mean.

FIG. 6. Absolute fluorescence emission of bean plants on two different days as a function of nitrogen treatment levels. The left panel shows data taken 43 days after planting; the right panel shows data taken 49 days after planting. The data points represent a mean of measurements in each fluorescence band for six plants for each nitrogen treatment level; the error bars represent one standard deviation of the mean. The results have not been normalized to the total leaf area in the field of view of the PFS共note: at 700 nm, 1 mW⬃6 nmol of photons兲.

(8)

one day to the next were caused by stress-induced changes in photosynthetic activity in PSII.

IV. DISCUSSION

In conclusion, we have successfully designed, built, and demonstrated a robust remote passive sensor for sunlight- induced plant fluorescence which is operates in two spectral bands共690 and 760 nm兲. It is capable of providing absolute measurements of fluorescence at the single leaf, whole plant, and canopy levels. It contains a minimum number of moving parts and runs with low-power consumption 共⬃75 W兲. The plant fluorescence sensor is fully capable of obtaining in vivo measurements of whole plant fluorescence under conditions found in the field as well as in the laboratory. Furthermore, its reliance on solar irradiation makes it ideal for canopy- level measurements in forests, agricultural settings, and grassland environments.

Data from a study of bean plants subjected to a range of nitrogen treatment are consistent with those achieved or pre- dicted with other passive fluorescence instruments.^19,20,22 Absolute plant fluorescence levels were found to inversely correlate with nitrogen treatment level—i.e., plants under stress showed significant increases in fluorescence intensity, especially in the red共690 nm兲band associated with emission from photosystem II. As a result, canopy-level measurements of the stressed plants exhibited large increases in R/FR共690 nm/760 nm兲 compared to plants not so stressed. This trend can be attributed to two factors. First, lower chlorophyll content in stressed plants leads to less self-absorption of 690 nm fluorescence emission 共chlorophyll does not absorb 760 nm radiation兲, and thus higher observed R/FR. Second, the intensity of the fluorescence emission from photosystem II共re- sulting in emission at 690 nm兲 reflects the physiological health of the plant, independent of chlorophyll content. The contribution of the latter was directly observed; stressed plants displayed statistically significant increases in R/FR over time without an accompanying change in chlorophyll content. Thus, the deployment of steady-state solar-excited plant fluorescence sensors offers the possibility of detecting plant stress well before visible changes in plant health are discemible.

ACKNOWLEDGMENTS

The major portion of this work was supported by the National Aeronautics and Space Administration under the Small Business Innovation Research Program. Additional support was provided by the Department of Energy 共also under the Small Business Innovation Research Program兲and internal funding by Aerodyne Research, Inc. The authors thank Dr. Gregory Carter of the NASA Stennis Space Center and Dr. Herman Scott of Aerodyne Research for their con- structive comments and advice.

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