GB2380790A

GB2380790A - Variation of led optical power and photosynthetic fluorometers

Info

Publication number: GB2380790A
Application number: GB0116779A
Authority: GB
Inventors: Neil Reeves Baker; Kevin Mark Oxborough; John Keith Bartington
Original assignee: University of Essex Enterprises Ltd
Current assignee: University of Essex Enterprises Ltd
Priority date: 2001-07-10
Filing date: 2001-07-10
Publication date: 2003-04-16
Anticipated expiration: 2021-07-10
Also published as: GB0116779D0; GB2380790A9; GB2380790B

Abstract

A method of varying the optical power output from a light emitting diode, 14 primarily for use in the irradiation of a chlorophyll-containing specimen to determine the photosynthetic characteristics of the specimen by measuring the fluorescence thereof when irradiated by the light from the diode. The spectral output of the diode is maintained substantially constant despite varying the illumination power, by driving the diode with a pulse-width modulated power waveform, the duty cycle of the waveform being variable to control the output power of the LED. Also described is a chlorophyll fluorometer employing an illumination LED for irradiating a specimen, where the power output of the LED is varied whilst maintaining a constant spectral output.

Description

VARIATION OF LED OPTICAL POWER AND PHOTOSYNTHETIC FLUOROMETERS This invention relates to methods of and apparatus for varying the optical power output from a light-emitting diode whilst maintaining substantially constant the spectral output of the light-emitting diode. Further, this invention relates to a method of operating a chlorophyll fluorometer utilising a light- emitting diode as an optical source, the power output of the source being controlled by the method aforesaid. The invention also relates to a chlorophyll fluorometer per se.

In its broader aspects, this invention may find application in many branches of science and technology, insofar as it provides both a method of and apparatus for varying the optical power output from a light-emitting diode (LED) while maintaining substantially constant the spectral output thereof.

However, in its preferred aspects, this invention is concerned with the provision of a light source for a chlorophyll fluorometer and methods of operating the same. In the following, the invention will hereinafter be described exclusively with reference to such a fluorometer though it is to be understood that in its broader aspects this invention is not to be regarded as limited to this particular application.

When exposed to incident light, many materials fluoresce. Analysis of the fluorescence may provide useful information about the underlying chemical, photoelectric and physiological processes. An instrument for performing such analysis is commonly known as a fluorometer. Such an instrument generates light of the required wavelength or band of wavelengths required to excite fluorescence in the sample under test and measures the light emitted from the

sample by the fluorescent materials present therein. Fluorometers are used in a wide variety of environmental, industrial and biological applications, including chlorophyll measurement, water quality analysis, RNA, DNA and protein quantitisation, histamine analysis, vitamin assays, afflatoxin analysis, antibiotic sensitivity, bacterial viability and blood analysis. The invention is primarily concerned with chlorophyll measurement though is not limited thereto.

Chlorophyll fluorescence stems from the fact that when exposed to a photosynthetically active radiation (PAR), the chlorophyll a (Chi a) contained within plants and other oxygenic organisms fluoresces. The analysis of the resultant Chi a fluorescence can provide useful information about the underlying physiological process, but to achieve meaningful results, it is essential that the level of illumination and the illumination spectrum or selected spectra are stable. The actinic light sources that are employed with many existing fluorometers suffer from changes in spectral characteristics with changes in light output. This can complicate the interpretation of fluorescence data and, in some situations, can make fluorescence data uninterpretable.

The characteristics of LED light sources are well known and understood, and such sources have consequently been used in chlorophyll fluorometers, particularly in view of their constant and repeatable spectral characteristics, at a given power. However, it is found that the spectral content of the output from a LED source varies significantly as the electrical power supplied to the LED, and so the optical output from the LED, is varied. In turn, this can lead to the requirement for very complex calibration and compensation techniques for a fluorometer, in order that consistent results can be obtained for different levels of illumination, which are required in order fully to exploit the photosynthetic

characteristics of a single sample under test, as well as for comparison between different samples.

As a LED appears to be a suitable power source for a chlorophyll fluorometer since it can be designed to emit light only within a very narrow frequency band, which may be optimised for chlorophyll fluorometry, this invention stems from research into ways of varying the optical power emitted by at least one LED by adjustment of the power source therefor, whilst maintaining substantially constant the spectral characteristics of the LED.

According to a first aspect of the present invention, therefore, this invention provides a method of varying the optical power output from a LED for use in the irradiation of a chlorophyll-containing specimen to determine photosynthetic characteristics of the specimen by measuring the fluorescence of the irradiated specimen, which method maintains substantially constant the LED spectral output, comprising connecting the LED to an electrical power source, and then supplying from the source a pulse-width modulated power waveform to the LED to cause the illumination thereof, the duty cycle of the waveform being variable to control the output power of the LED.

Though pulse-width modulation is a known and accepted way of controlling the power supplied to a component, including an incandescent light source, its use with one or more LEDs to control the optical source for a chlorophyll fluorometer gives rise to several unexpected but significant advantages. In particular, by controlling the optical power output in this way, it has been found that the spectral output is substantially constant, irrespective of the optical power output of the LED over a wide range of optical powers. In addition, it is found that the use of a pulse-width modulated waveform gives rise

to advantages during calibration of the fluorometer to maximise the dynamic range of the fluorometer. In part, this arises because changing the duty cycle of the pulse-width modulated drive signal changes the light output from the LED in direct proportion thereto, over a significant range of drive signal duty cycles. For example, a doubling of the duty cycle doubles the light output and this much facilitates the setting-up and calibration procedures.

Most preferably, the pulse-width modulated waveform supplied to the LED is a periodic substantially square-wave waveform. Further, that square waveform should have alternate on-periods where a constant current is supplied to the or each LED and off-periods where no current is supplied. In this way, the maximum power of the pulse-width modulated waveform may be substantially constant during the on-period, so leading to a constant spectral output, though the average power will vary with the selected duty cycle.

Spectral variation may be minimised by maintaining the transition time of the square-wave, between an off-period to an on-period and between an on-period to an off-period. This transition time should be made as short as possible, and in particular several orders of magnitude smaller than the shortest on-period, so as to have an insignificant effect on the overall spectral stability.

In a typical system, transition times of around 30ns may be achieved, with the on-time ranging from 1 pus up to perhaps 200Jls. This gives a 200: 1 optical power range. The proportion of the total transition times for one complete cycle will depend upon the off-time, but may range from about 5% with a minimum on-time of 1 Jls, up to about 0.03% with a 200ps on-time, but in each case with an off-time of about 1 ps. The maximum off-time should not

exceed a few hundred lis, else there will be an undesirable effect on the photosynthesis process giving rise to the fluorescence signal.

According to a second aspect of this invention, there is provided a method of operating a chlorophyll fluorometer comprising a specimen holder, illuminating means for the specimen, and fluorescence analysis means arranged to collect fluorescent light emanating from a held sample, wherein the illuminating means includes at least one LED and an electrical power source therefor arranged to provide a pulse-width modulated power waveform, in which method the optical power output of the LED is varied by varying the duty cycle of the waveform while maintaining substantially constant the spectral output of the LED.

In yet another aspect of this invention, there is provided a chlorophyll fluorometer comprising a specimen holder, illuminating means for the specimen and fluorescence analysis means arranged to collect light emanating from a held specimen under test, wherein the illuminating means comprises the combination of a LED and an electrical power source arranged to provide a pulse-width modulated square-wave waveform to the LED, whereby the duty cycle of the waveform may be adjusted to control the optical power of light from the LED. Preferably, the illuminating means comprises at least one planar array of a plurality of LEDs connected in parallel, in series or in series/parallel, and arranged to direct light to a held specimen.

The fluorescence analysis means preferably comprises a video camera arranged to collect fluorescent light from a specimen under test and to generate a Chl a fluorescence signal which is supplied to a computer for storage therein

and analysis by a suitable program running on the computer. The computer may then control the pulse-width modulated waveform in order to give an appropriate photosynthetically active photon flux density (PPFD) for illuminating the specimen.

An aim in setting up the fluorometer of this invention is to optimise the available dynamic range of the video camera, by suitable adjustments to the PPFD, the camera gain and camera shutter speed. This may be achieved by having the computer run an appropriate program to analyse the image produced by the camera, by counting pixels of the image above and below certain predefined intensity values, aiming to achieve a pixel count falling within a predefined range. In this way, the combination of PPFD incident at the time of imaging, camera gain and camera shutter speed can be set fully to exploit the dynamic range of the camera.

By way of example only, one specific embodiment of a chlorophyll fluorometer constructed and arranged in accordance with the present invention and having a LED array as its light source, which array is also controlled in accordance with the present invention, will now be described in detail, reference being made to the accompanying drawings, in which :- Figure1 is a diagrammatic illustration of the important components of the fluorometer ; Figure 2 is a block diagram of the control circuit for the array of LEDs used in the fluorometer ; Figures 3A, 3B and 3C are timing diagrams for the power supply which energises the array of LEDs;

Figure 4A illustrates the spectral distribution of a LED the optical output power of which is controlled by voltage modulation; Figure 4B illustrates the spectral distribution of the LED when the optical output is controlled by pulse-width modulation ; and Figure 5 shows the variation in the optical power output of an LED when powered by a pulse-width modulated drive current the duty cycle of which varies.

Referring initially to Figure 1, there is shown diagrammatically an embodiment of chlorophyll fluorometer of this invention. A sample (not shown) under test is supported on a platen 10, below a video camera 11 arranged to scan an image of the sample on the platen. The image obtained from the camera is fed to a computer 12 where that image is analysed by the computer running an appropriate program. The computer supplies a control signal to a pulse-width modulation power supply module 13, which in turn supplies power to an array 14 of LEDs to cause illumination of those LEDs and so of the sample on the platen 10. Typically, the array will have 100 LEDs arranged in a 10x10 matrix and though only a single array is shown in Figure 1, the fluorometer may have several such arrays, to give uniform illumination of the sample on the platen. In this case, all of the arrays should be driven with the same control signal so as to provide simultaneous illumination. In the alternative, some of the LEDs of one or more of the arrays may selectively be turned off to permit adjustment of the lighting.

The LEDs used in the or each array are selected to have a suitable spectrum for the analysis being performed. For Chi a analysis, the spectrum of the LEDs may be in the region of 460nm to 480nm for optimum fluorescence

results though other spectra may be chosen for particular samples or for specific purposes. The power supply should have the ability to turn on and off the supply of current to the arrays sufficiently rapidly that the transition time taken for a LED to turn on or to turn off is insignificant in relation to the minimum required period to achieve a specific mean level of lighting for the sample. Typically, illumination of the sample of at least 1 pus is required though the illumination may continue for as long as 200s or even longer. By contrast, the turn-on and turn-off transition times for the array may be in the region of 30ns.

Figure 2 shows a block diagram of the power supply arrangement for driving the array 14 of 100 individual LEDs 15. The computer 12 generates three digital pulse outputs 16,17 and 18, all three of which are supplied to a control circuit 19. Output 16 is used to select either output 17 or output 18 within control circuit 19, depending upon the mode within which the fluorometer is to operate. Outputs 17 and 18 are both pulse trains with different duty cycles and periods, determined by the computer, and are used to control the average amount of light produced by the (or each) array 14 of LEDs. The power for driving the LEDs is derived from a high current constant voltage power source 20; the array is energised when current is switched on by an n-channel powerFET 21, the gate signal for which is derived from a FET driver circuit 22, itself driven by the selected pulse train 17 or 18, from the control circuit 19. When the power-FET 21 is on, p-channel power-FET 23 is off. Conversely, when power-FET 21 is off, power-FET 23 is held on to ensure that any residual capacity of charge in the LED array 14 does not increase the turn-off time, by in

effect shorting-out the array. Though not shown in Figure 2, a 12-volt boost power supply is derived from the high current power source 20, to supply up to 1.5A to the driver circuit 22, for driving the gates of the FETs as required.

Figures 3A, 3B and 3C are timing diagrams for the arrangement described above. As can be seen from Figure 3C, the rise time and fall time for the essentially square-wave pulse-width modulation signal is approximately 30ns, this being the fastest turn-on and turn-off time achievable with conventional electronics, without employing highly complex and expensive circuit construction techniques. By contrast, the minimum on-time (Figure 3A) for the pulse-width modulation signal is at least one 1 pus, though the on-time may be extended to up to 200s (Figure 3B), unless continuous illumination is required.

The interval between the on-pulses must be short, and typically no more than a few hundred Vs. Long dark periods would result in undesirable fluctuations in the redox state of the electron carriers in the photosynthesis process and also in the size of metabolite pools. A high-duty cycle is therefore composed of a long on-time combined with a short off-time. A low-duty cycle is composed of a very short on-time combined with an off-time of no more than a few hundred Vs. The minimum duty cycle may be as low as 0.2% and the maximum duty cycle just short of 100%, unless continuous illumination is required-that is to say, 100% duty cycle with no off-times.

The above may be achieved by varying period for the pulse-width waveform in conjunction with variations in the duty cycle. A low-duty cycle will

require a short period to ensure the off-time is not too long, but a high-duty cycle may utilise a longer cycle period.

Figure 4A illustrates the spectral distribution of an LED when the light output is varied by way of voltage modulation. As can be seen, the centre wavelength emitted from the LED varies significantly as the power output is varied by adjusting the drive voltage to the LED. By contrast, Figure 4B illustrates the spectral distribution of the same LED when the light output is varied by way of pulse-width modulation of the drive current to the LED, with a constant power during each on-period for the LED. As can be seen, the spectral distribution has essentially a constant centre wavelength irrespective of the power.

Figure 5 plots the power output (intensity) of the LED against the duty cycle of the drive current for the LED. Over a wide range of duty cycles, there is a linear relationship between the duty cycle and the optical power output from the LED. This can greatly facilitate the calibration and setting-up procedures for the fluorometer of Figure 1, since a doubling or halving of the duty cycle (within the linear range) will double or halve the optical power output.

The illumination drive arrangements for the fluorometer described above enables the simple operation of the device over a wide range of samples.

Further, the incident PPFD during imaging can be adjusted to suit determined changes in the prevailing Chi a fluorescence signal characteristics, so optimising the dynamic range of the camera 11. The amount of useful information that can be derived from the Chi a fluorescence measurement is greatly increased if the Chi a fluorescence signal can be recorded over a wide range of continuous incident PPFDs, from the equivalent of several times full

sunlight down to complete darkness. A very low incident PPFD may be required to satisfy the biological requirements and a high incident PPFD to maximise the signal-to-noise ratio of the fluorometer. The setting-up procedures for the fluorometer described above may permit the optimisation of the exposure length and incident PPFD during recording of the Chi a signal to provide the most desirable combination of incident PPFD and fluorescence signal intensity. Where the continuous incident PPFD is zero or low, integrated measurements of Chi a fluorescence may be made with long exposures, while highly resolved images of Chi a fluorescence may be taken during long exposures with a higher PPFD. A long exposure time at low PPFD offers a biological advantage over the delivery of the same number of photons but at a higher PPFD, with a short exposure time. This advantage outweighs the consequent disadvantage of a decrease of the signal-to-noise ratio.

At high levels of continuous incident PPFD, images for the integrated Chi a fluorescence signal and highly resolved images of Chi a fluorescence signal are both taken at the prevailing PPFD. The fluorescence signal is brought within the upper end of the dynamic range of the camera by setting appropriate values for the exposure length and camera gain. These values can be determined using a calibration image which is produced at the start of a series of measurements when the sample is in a known biological state. The prevailing integrated fluorescence signal is normalised to take into account the different combinations of exposure length and camera gain, using values derived from an automated calibration routine, controlled by the computer.

Claims

CLAIMS 1. A method of varying the optical power output from a light-emitting diode (LED) for use in the irradiation of a chlorophyll-containing specimen to determine photosynthetic characteristics of the specimen by measuring the fluorescence of the irradiated specimen, which method maintains substantially constant the LED spectral output, comprising connecting the LED to an electrical power source, and then supplying from the source a pulse-width modulated power waveform to the LED to cause the illumination thereof, the duty cycle of the waveform being variable to control the output power of the LED.
2. A method as claimed in claim 1, wherein the pulse-width modulated waveform supplied to the LED is a periodic substantially square-waveform.
3. A method as claimed in claim 2, wherein the square-waveform has alternate on-times where current is supplied and off-times where no current is supplied.
4. A method as claimed in claim 3, wherein the maximum power of the pulse width modulated waveform is substantially constant, during the on-times of the square-waveform.
5. A method as claimed in claim 3 or claim 4, wherein the transition time of the square-waveform, between the on-times and off-times, is maintained at not more than about 5% of the instantaneous on-time of the square-waveform.
6. A method as claimed in any of claims 2 to 5, wherein the total periodic cycle time of the pulse-width modulated waveform for each cycle of operation is maintained substantially constant for a predetermined range of optical power outputs, achieved by varying the duty cycle of the waveform.

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7. A method as claimed in any of the preceding claims, wherein the power supply is arranged to supply a selected one of a plurality of pre-set waveform cycle times and a suitable cycle time is selected having regard to the required duty cycle of the waveform to be supplied.
8. A method as claimed in claim 6, wherein a different waveform cycle time is selected for small duty cycles to give a relatively low optical power output than for large duty cycles to give a greater optical power output..
9. A method as claimed in any of the preceding claims, wherein the duty cycle of the waveform is variable between 0.5% and 100% of the total cycle time.
10. A method as claimed in any of the preceding claims, wherein the duty cycle of the waveform is automatically controlled by a computer program monitoring the fluorescence of a sample illuminated by light from the LED.
11. A method as claimed in claim 10, wherein the fluorescence is observed by a video camera the output of which is monitored by the computer program, and the duty cycle of the waveform is selected to maximise the use of the available dynamic range of the video camera.
12. A method as claimed in claim 11, wherein the computer program additionally controls the operation of the video camera.
13. In combination, a LED and an electrical power source connected to supply current to the LED, the power source being arranged to supply a pulsewidth modulated power waveform to the LED to cause the illumination thereof, the power source having an adjustable control means to permit variation of the duty cycle of the waveform thereby to control the optical output power of the LED.

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14. A method of operating a chlorophyll fluorometer comprising a specimen holder, illuminating means for the specimen, and fluorescence analysis means arranged to collect light emanating from a held sample, wherein the illuminating means includes a LED and an electrical power source therefor arranged to provide a pulse-width modulated power waveform, in which method the optical power output of the LED is varied by varying the duty cycle of the waveform whilst maintaining substantially constant the spectral output of the LED.
15. A method as claimed in claim 14, wherein the optical output of the LED is varied in accordance with a method as claimed in any of claims 2 to 12.
16. A method as claimed in claim 14 or claim 15, wherein the illuminating means comprises a plurality of LEDs electrically connected and arranged in a substantially planar array, the array of LEDs being simultaneously driven by the pulse-width modulated power waveform to vary the optical power output thereof.
17. A method of operating a chlorophyll fluorometer as claimed in any of claims 14 to 16, wherein the fluorescence analysis means includes a video camera collecting fluorescence light from the sample and producing a pixelbased image thereof, and the output of the camera is analysed by counting the number of pixels of the image at or within a pre-set luminosity range, the optical output of the illuminating means then being varied dependent upon the pixel count to maximise the dynamic range of the camera.
18. A chlorophyll fluorometer comprising a specimen holder, illuminating means for the specimen, and fluorescence analysis means arranged to collect light emanating from a held specimen under test, wherein the illuminating

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means comprises the combination of a LED and an electrical power source according to claim 13.
19. A chlorophyll fluorometer as claimed in claim 17, wherein the illuminating means comprises a plurality of LEDs electrically connected and arranged in a substantially planar array.
20. A method of varying the optical power output from a light-emitting diode (LED) whilst maintaining substantially constant the spectral output and substantially as hereinbefore described with reference to Figure 2 of the accompanying drawings.
21. A method of operating a chlorophyll fluorometer and substantially as hereinbefore described with reference to the accompanying drawings.
22. A chlorophyll fluorometer substantially as hereinbefore described with reference to and as illustrated in Figures 1 and 2 of the accompanying drawings.