stable 2.5 V signal which is applied to a voltage divider, resistor R10 and potentiometer R11, to generate a constant voltage OUT signal ranging between 0 and 2.5 V. This signal i s then buffered bv a voltage follower (IClC) andapplied t i t h e second inout of the summing amdifier. f i e summing ampGfie; sums this constant signal with the inverted signal from the photodiode amplifier, then amplifies the summed signal. Thus, potentiometer R11 serves as an adjustable offset control for the output from the fluorescence detector. A calibration mode on the fluorescence detector provides a check for amplifier stability durFigure 2. Circuit Diagram of the Detector Amplifier. Unless otherwise noted all resistors are 114 W. See ine a series of measurements. te? for descriptive details. components IClD, R7, R8, SW1, and SW2 compose the calibration circuitry. Switch SW1 selects the measuring mode The LED must have uniform output, i.e., smooth, rapid (photodiode amplifier output is input to the summing amrise to a stable level of continuous irradiation. This can be plifier) or calibration mode (constant reference signal is best tested after several hours of LED "burn-in" at its substituted for the photodiode output) of the detector. specified maximum power. Testing can be accomplished Switch SW2 selects a constant reference signal of either using the fluorescence detector (described below) with its zero volts (ground) or 0.5 V that is generated from the 2.5output connected to an oscilloscope. A reflective sample is V reference via components IClD, R7, and R8. The gainand used, and stability of the light output from the LED is offset of the detector can be verified by selecting the caliassessed using both the flash mode and the continuous brationmode withSW1 and connecting the detector output mode to power the LED. to a voltage measuring device. Voltage output (VJ with SW2 switched to ground (V,) and the voltage output (Vz) Fluorescence Detector Amplifier with SW2 switched to the reference (V,d can he used to Detection of the weak chlorophyll fluorescence from a verify the gain and offset for a particular configuration of sample irradiated by the LED is achieved by amplifying the the instrument, since: GAIN = (Vz-V1)/0.5, and OFFSET = output from a TIL413 photodiode. Characteristics of this (0.5 x Vl)/(V2-Vl). This calibration check is useful to conphotodiode include temperature stability, 100 ns rise time, firm the consistency of the fluorescence detector output linear output, and high sensitivity to light wavelengths during a series of measurements where fluorescencechargreater than =700 nm. The amplifying circuit for the fluoacteristics of different samples are compared. rescence detector diagrammed in Figure 2 amplifies the output signal of the photodiode and provides controls for Optical System of the Detector signal offset and calibration. The dual power DC source for the amplifier (not shown) should be in the range 9-15 V for Basic design for actinic irradiation of a leaf sample and V+ and -9 to -15 V for V-. A weu-regulated, filtered, dual detection of the fluorescence from the leaf surface is shown voltage, line-operated voltage supply that provides several in Figure 3. Actinic irradiation at a fured 60" angle to the tens of mAcan be used. This supply must be well isolated leaf surface and detection of emitted fluorescence normal from the amplifying circuit and from the LED power supply to leaf surface minimizes measurement of light re~- the - - ~ to minimize AC noise. Alternatively, two 9 V transitor flected from the sample surface. (Because of reabsorption batteries can be used. Grounding of the LED controller of fluorescence by the dense chlorophyll array in a leaf, circuit to the LED mount is necessary to reduce AC noise measured chlorophyll fluorescence is probably limited to in the fluoresceuce detector output signal. that emitted from the cells near the irradiated surface.) The voltage signal output of the photodiode (Dl) is iniLight guides of Pyrex glass rod sheathed within the brass tially amplified by an inverting amplifier composed of tube mounts eliminate scattered light and provide a heat IClA, R1, R2, R3, and Dl. During construction of the sink for the LED. The actinic 665 nm irradiance at the circuit, minimal common-modeamplificationis achieved as sample plane is 17 W m-2 or 0.85 W m-%with the LED follows: D l is shorted, and R3 is adjusteduntil the amplifier powered on "high" (forward current = 30 mA) or on 'low" output is minimal. After this initial adjustment, no further (forward current = 4.0 mA), respectively. Fluorescence irchange of R3 should be necessary. radiation is passed through a cut-off filter disc of far-red plexiglassfilter (CBSFar Red 750, from Carolina Biological The amplified photodiode signal is normally applied to Supply Company, Burlington, NC) before detection by the one of two inputs on a second amplifier that functions as a photodiode. Light transmittedby the filter is less than0.1% variable gain, inverting, summing amplifier. Integrated T - at 680 nm and 73% T a t 750 nm: the transmission circuit IClB and resistors R4, R5, and R6 compose this spectrum is similar to that of the photodiode casing. The amdifier. Potentiometer R6 controls the gain to a maxientire assembly is mounted in a n appropriately machined mum of approximately -25X. Output from this amplifier, housing that provides for fixed sample mounting geometry the fluorescence detector simal, is connected to a recording device, e.g., a chart recorder. and a light-proof enclosure of the optical components. A removable cap allows convenient sample changes but The second input to the summing amplifier is from the appresses the leaf sample gently over the aperture of the offset control circuit. Resistor R9 and diode D2 generate a ~~~
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Journal of Chemical Education
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Time Fig~re4.Ch oropnyllfJorescence nauct on in leavesof Wgnaradiata. Mat~re,fully-expandedleaf (A), comparably ageo, out senescenc ng eaf (8).Tne mat~releaf naa 2.5 t rnes more cn orophyll. C J N (61 ~ has been displaced along the time axis for clarity. Intensity level of the constant fluorescence (ophyll content of the senescent leaf, they also suggest that thc photosynthetic apparatus of the senescent leaf had been altered. The different F, values indicate differences in the photirhemical actirity of the PSI1 complex (2);the different patterns of fluoresce&e decay from Fp indicate changes in the chloroplast that affect the non-photochemical quenching of fluorescence, i.e., chloroplast capacities for proton pumping and ion exchange. - . .~hosohorvlatiouof antenna chloro~hvllmolecules, and biocLemi&l utilization of absorbed Lg6t energy for carbon reduction (2.4. 6).Results from such measurements can form the baiis for further investigations of changes in photosynthetic competence that occur in response to herbicides, environmental stress, or plant development. A
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Literature Cited 1.Papageom$m", G.h Bioenergetics ofPhotasynthesis; Conndjee, Ed.; Academic Press: New Y a k , 1975;Chap.6. 2. Krause. G.H.: W e i s E. Phatosvr. Res. 1984.5.139-157. 3.Smillie.. R.M.:Hethelinetan. S. E. Plant Phvaiol. 1983.72.1043-1050. . 4.Toiuonen, P.;Vidaver. W. Plant Phyiol. 1988,86,74&748. 5. Schreiber, U.Photasyn. Res 1983,4,361373. 6. Bruce, D.:Vidaver,W.: Colbaru, K:and Popovie, R.Plant Physiol. 1983,73,386888.
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Volume 68 Number 11 November 1991
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