Patent Document

CLAIM OF BENEFIT TO RELATED APPLICATION 
   This application is a continuation application of U.S. Nonprovisional patent application Ser. No. 11/303,446 filed Dec. 15, 2005 now U.S. Pat. No. 7,301,158, entitled “Method and Apparatus for Measuring Active Fluorescence,” which is incorporated herein by reference. U.S. Nonprovisional patent application Ser. No. 11/303,446 claims benefit to U.S. Provisional Patent Application 60/637,478 filed Dec. 15, 2004, entitled “Method and Apparatus for Measuring Active Fluorescence.” 

   BACKGROUND OF THE INVENTION 
   The use of fluorescence to measure the photosynthetic activity of organisms has been an accepted method in the scientific community for many years. Measuring fluorescence as a function of photosynthetic activity, compared to other methodologies, is fast, easy to use, and requires relatively low cost instrumentation. The majority of fluorometers in use today are classified as passive fluorometers and are used to measure the total biomass of photosynthetic organisms. Passive fluorometers supply a constant source of light of a specific wavelength and measure the light output from the sample at a different, typically longer, wavelength. In order to measure sample levels as low as possible, passive fluorometers typically use as bright a light as possible. 
   A known drawback to using passive fluorescence is that the fluorescent output of a sample can vary due to several influences not related to biomass of the photosynthetic organisms. For example, an organism&#39;s fluorescent light output will vary depending on the ambient light condition of its environment. In addition, the source light used by the fluorometer can affect the organism being measured. 
   Active fluorescence overcomes these issues by using flash stimulated fluorescence. An example of active fluorescence is disclosed in Moll, U.S. Pat. No. 4,650,336. Moll describes a method and device to measure photosynthetic activity using variable fluorescence. Moll uses one lamp to provide constant-level light to bring about continuous, steady state fluorescence of a plant, and a flash lamp to provide a flash of light to bring about a transient fluorescence of the plant. 
   Another active fluorescence technique is described in Kolber et al., U.S. Pat. No. 4,942,303 (“Kolber I”). Kolber I describes a “pump and probe” technique that uses a low intensity “probe” flash to measure fluorescence before and after a bright “pump” flash to measure the change in fluorescence. 
   Another active fluorescence technique is described in Kolber et al., U.S. Pat. No. 5,426,306 (“Kolber II”). This technique, known as fast repetition fluorescence, uses a series of fast, repetitive flashes at controlled energies to incrementally effect photosynthetic processes. 
   Another active fluorescence technique is described in Kolber et al., U.S. Pat. No. 6,121,053 (“Kolber III”). Kolber III describes a multiple protocol fluorometer which allows a significant amount of control over the duration, frequency, and power of the flashes. 
   The trend in active fluorometer development, as can be seen in the references, has been towards providing researchers with progressively more control over the active fluorescence protocol with the goal of providing increasingly detailed information of the photosynthetic process. While this is a laudable goal, it has led to the development of increasingly complex and costly instruments. With limited research budgets, many researchers cannot afford the instruments currently available. 
   The components used by current active fluorometers are one reason for their high costs. Bright light sources such as the flash lamps used in Kolber I and Kolber II require a large amount of energy to work properly, and require expensive support circuitry to supply the currents they need. Moving to solid state LEDs as used in Kolber III is a step in the right direction, since they require less power and less support circuitry. But Kolber III uses a large array of LEDs driven above their nominal currents, again requiring a significant amount of energy. The use of bright light sources is due to the weak fluorescent response of algae in water as compared to solid samples (e.g., a leaf). Using a bright light helps maximize the response to improve detection limits. 
   Another source of large material cost is the detector for measuring the light emitted by the sample. Because the emitted light is relatively dim for algae in water, the photodetector in the above references has been a photomultiplier tube (“PMT”). A PMT is a vacuum tube with special elements to convert a detected photon to an electrical current which is then amplified internally before being provided to outside circuitry for further signal processing. PMTs are inherently expensive due to their specialized nature, many are built by hand. In addition, they require high voltage sources to operate (e.g., up to 1000 volts) which can also be expensive. Due to their construction, PMTs are fairly fragile. Not only are they encased in glass under vacuum, but the internal elements are small metal plates that are carefully aligned. PMTs therefore do not handle shock very well. In addition, PMTs are typically physically large. This makes it difficult to place them near the sample. Kolber II uses optics such as lenses and collimators to collect emission light from the sample and provide it to the PMT, again increasing components, complexity, and cost. 
   Further costs have been added due to the emphasis on increasing control, data acquisition, and data analysis to calculate the many parameters of photosynthesis. This requires the use of more powerful, and hence more costly, internal computers. 
   The light sources, detectors, and computers of the current designs are all large and require a significant amount of power. This leads to large enclosures and large power sources, again increasing costs. 
   In addition to limited or decreasing budgets, there is a trend among researchers towards the deployment of multiple sensors in situ in various locations collecting data in real time to give a broader view of the health of a body of water. Often these instruments are deployed in fixed locations (e.g., a pier) and are left unattended to operate for significant periods of time (e.g., one month). An ideal instrument would have a low enough cost to allow the purchase of multiple units and would have low enough power consumption to operate on a battery for the necessary period of time. In addition, many studies only require the information that can be provided by a basic active fluorometer. All of these factors lead to the conclusion that there is a need for a small, low cost, low power, and reliable active fluorometer. 
   SUMMARY OF THE INVENTION 
   Some embodiments of the invention provide an apparatus for measuring active fluorescence in liquid samples by using solid-state components. The use of solid-state devices dramatically lowers the cost, size, and power consumption of active fluorescence while improving the ruggedness and reliability. The smaller size of the solid-state devices allows them to be placed very close to the sample. This maximizes the amount of light the sample receives from the light sources and allows efficient collection of the resulting emitted light using simple and low cost optical components. 
   In some embodiments, the apparatus (1) uses either a single LED or a few LEDs (4 or fewer) that are modulated to provide a measuring light source and (2) uses a small number of LEDs (12 or fewer) to provide a saturating light source. Also, the apparatus includes a photodetector that is a photodiode. The apparatus includes amplification circuitry associated with this photodiode. This amplification circuitry is synchronized to both the modulating light source and the analog to digital conversion. The apparatus in some embodiments further includes a small, low power microcontroller to control the light sources and to read, report, and/or record the output from the photodiode and its associated circuitry. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The novel features of the invention are set forth in the appended claims. However, for purpose of explanation, several embodiments of the invention are set forth in the following figures. 
       FIG. 1  illustrates a top view of the components of the fluorometer. 
       FIG. 2  illustrates a block diagram of the electronic components of the fluorometer 
       FIG. 3  illustrates a schematic of the detection circuit of the fluorometer. 
       FIG. 4  illustrates a graph of the timing of the sampling LED and the measuring electronic components. 
       FIG. 5  illustrates a flow chart of the process to obtain information on the photosynthetic process. 
       FIG. 6  illustrates a protocol for the fluorometer to determine certain parameters of the photosynthetic process. 
       FIG. 7  illustrates another protocol for the fluorometer to determine certain parameters of the photosynthetic process. 
       FIG. 8  illustrates yet another protocol for the fluorometer to determine certain parameters of the photosynthetic process. 
       FIG. 9  illustrates an alternative embodiment of the fluorometer. 
       FIG. 10  illustrates another alternative embodiment of the fluorometer. 
       FIG. 11  illustrates another alternative embodiment of the fluorometer. 
       FIG. 12  illustrates another alternative embodiment of the fluorometer 
   

   DETAILED DESCRIPTION OF THE INVENTION 
   In the following description, numerous details are set forth for purpose of explanation. However, one of ordinary skill in the art will realize that the invention may be practiced without the use of these specific details. In other instances, well-known structures and devices are shown in block diagram form in order not to obscure the description of the invention with the unnecessary detail. 
   I. Active Fluorometer 
     FIG. 1  conceptually illustrates a fluorometer having an optical configuration  100 . As shown in this figure, the optical configuration  100  includes a sample container  105 , a sampling light emitting diode (“LED”)  110 , an excitation filter  120 , a first optical lens  125 , a saturating LED  130 , a saturation excitation filter  140 , a second optical lens  150 , an emission filter  160 , a photodiode  170 , a first aperture  180  and a second aperture  190 . 
   In some embodiments, a water sample can be placed in the sample container  105 . While  FIG. 1  illustrates the sample container  105  to be square, in other embodiments, the sample container  105  can just as easily be round, rectangular, or other shapes depending on physical requirements. 
   The sampling LED  110  is oriented to shine into the sample water. Specifically, during the operation of the fluorometer, the sampling LED is modulated to provide a measuring light source.  FIG. 1  illustrates one sampling LED  110 . However, in other embodiments, there can be more than one sampling LED  110 . 
   In some embodiments, the excitation filter  120  is placed between the sampling LED  110  and the sample container  105 . Some embodiments that have more than one sampling LED have one excitation filter for each sampling LED. Other such embodiments might share one excitation filter among more than one sampling LED. While the LEDs in general produce a narrow bandwidth of light, adding the excitation filter  120  further reduces the bandwidth to the wavelength of interest and generally produces more satisfactory results. In other words, the excitation filter filters out wavelengths of light (emitted by the LED or LEDs) that are not of interest (i.e., that do not lead to the desired fluorescence effect). 
   In some embodiments, the lens  125  is placed between the sampling LED excitation filter  120  and the sample container  105  to focus the light from the sampling LED  110 . The aperture  180  collimates the light from the sampling LED  110  and helps to prevent stray light from shining directly into the photodiode  170 . 
   At least one saturating LED  130  is placed around the sample, with one or more excitation filter(s)  140 . During the operation of the fluorometer, the saturating LED  130  provides a saturating light source, while its associated excitation filter  140  filters out wavelengths of light (emitted by its LED) that are not of interest (i.e., that do not lead to the desired saturation effect). The embodiments that use multiple saturation LEDs might share one excitation filter among more than one saturation LED. 
   The saturating LED  130  is typically the same type of LED as the sampling LED  110 , though the saturating LED  130  may be driven at higher currents to give maximum light output. In some embodiments, the excitation filter  140 , if used, is typically the same type of filter as the excitation filter  120 .  FIG. 1  only shows one saturating LED  130 , however there can be as many as needed to fully saturate the water sample. In some embodiments, with a small sample volume, typically 12 or fewer saturating LEDs  130  and their associated excitation filters  140  are needed. A saturating LED  130  may be oriented in any direction, though it is usually advisable to avoid shining the LED directly at the photodiode  170 . 
   In some embodiments, the detector is the photodiode  170  that is typically oriented orthogonally to the sampling LED  110 . The photodiode  170  has an emission filter  160  to measure only the wavelength of interest. The lens  150  is optionally used in some embodiments to gather more light from the sample and focus the light on the photodiode  170 . The aperture  190  is again used to collimate the emission light and to prevent stray light from sampling LED  110  or saturating LED  130  from reaching the photodiode  170 . 
   The saturation operation of some embodiments provides light to the sample for a long enough period of time to cause the sample to be at its maximum fluorescence. 
   When the sampling LED  110  is on, it causes some living things in the water to fluoresce, but when the sampling LED is off, the photodiode  170  may still receive some light, either ambient light, or light from the saturating LED  130 , or fluorescence caused by the saturating LED  130 . The fluorometer distinguishes between fluorescence caused by these extraneous sources by measuring the light received by photodiode  170  when sampling LED  110  is on, and when it is off. The difference between these two measurements is the relative fluorescence of the sample. 
   As further described by reference to  FIG. 5  below, the system in some embodiments initially takes a measurement using just the sampling LED. This measurement defines the Fo level, and can be used for comparison to later measurements. The system then turns on the saturating LED  130  for some longer period of time, in order to cause the sample to reach its maximum fluorescence Fm, which the system measures using the sampling LED. The system then uses the measured Fo and Fm levels to measure the sample&#39;s yield, which is indicative of the health of the living things in the sample. 
   II. Circuitry for Active Fluorometer 
     FIG. 2  conceptually illustrates the electronic control system  200  of the fluorometer of some embodiments. This control system includes a microcontroller  205 , a sampling LED drive circuit  210 , a saturation LED drive circuit  215 , an amplifier  220 , a gain control circuit  230 , an analog switch  240 , a first hold circuit  250 , a second hold circuit  260 , and a differential amplifier  270  and a detection circuit  290 . 
   The microcontroller  205  for driving the sampling LED  110  and saturating LED  130 , and processing signals from the photodiode  170 . In some embodiments, the microcontroller  205  is a commercially available low cost microcontroller that includes a processor, a memory, digital inputs and outputs, an analog to digital converter, and a communicator for communicating with an external computer. While there are many low cost commercially available microcontrollers, some embodiments may use a dedicated microcontroller that is specifically designed for the fluorometer. 
   The microcontroller  205  is responsible for supplying signals that determine (1) when the sampling LED  110  turns on and off, (2) when the saturating LED  130  turn on and off, (3) what gain should be selected by the gain control circuit  230 , etc. The gain control circuit  230  will be further described below. A digital signal  209  from the microcontroller  205  controls the sampling LED drive circuit  210 . Similarly, a digital signal  214  from the microcontroller  205  controls the saturating LED drive circuit  215 . The LED drive circuits  210  and  215  are designed to supply a precise amount of current to the sampling LED  110  and saturating LED  130  respectively, and to turn on and off the sampling LED  110  and saturating LED  130  very quickly. 
   In some embodiments, detection circuit  290  (illustrated in more detail in  FIG. 3 ) includes the photodiode  170  that supplies a small signal  219  to the amplifier  220  which boosts the small signal  219  to a larger value. The output of the amplifier  220  goes to the gain control circuit  230 . The gain control circuit  230  is controlled by a digital signal  229  from the microcontroller  205 . The purpose of gain control circuit  230  is to further amplify the output of the amplifier  220  if needed so that the signal is at a level which can be read by the analog input of the microcontroller  205 . If the output signal of the amplifier  220  is small, then additional gain would be selected. If the output signal of the amplifier  220  is large, then no additional gain would be needed. 
   The output signal  239  from the gain control circuit  230  goes to the analog switch  240 . The analog switch  240  is controlled by the same signal  209  which drives the sampling LED circuit  210 . By switching in this manner, the output signal  249  of the analog switch  240  will always present the signal from the photodiode  170  when the sampling LED  110  is on. Likewise, the output signal  259  of the analog switch  240  will always present the signal from the photodiode  170  when the sample LED  110  is off. The hold circuits  250  and  260  are needed to hold the signal levels of the output signals  249  and  259  respectively, since the output signals  249  and  259  are not constantly present due to the continuous switching of the analog switch  240 . The hold circuit  250  will hold the amplified signal from the photodiode  170  when the sampling LED  110  is on. The hold circuit  260  will hold the amplified signal from the photodiode  170  when the sampling LED  110  is off. 
   The output signal  268  of the hold circuit  250  becomes the signal to the positive input of the differential amplifier  270 . The output signal  269  of the hold circuit  250  becomes the signal to the negative input of the differential amplifier  270 . The output signal  204  of the differential amplifier  270  is the amplified difference between the output signals  268  and  269 . 
   As mentioned above in the description of  FIG. 1 , the fluorometer of these embodiments measures the effect of the sampling LED  110  by subtracting out the level of light found when the sampling LED  110  is off. In some embodiments, the differential amplifier  270  is used to perform ambient light rejection. Ambient light is light in the sample&#39;s environment that is not a function of fluorescence. As such, it is an unwanted background signal. By measuring the output of the photodiode  170  when the sampling LED  110  is off, the ambient light is determined and subtracted from the measured light when the sampling LED  110  is on, thus providing a measure of fluorescence which is not affected by background illumination (e.g., ambient light). In some embodiments, where measurements are taken while the saturating LED  130  is turned on, the differential amplifier also serves to reject that portion of the fluorescence that is caused by the saturating LED  130  and not by the sampling LED  110 . An analog to digital circuitry contained in the microcontroller  205  uses the output signal  204  from the differential amplifier  270  and converts it to a digital value which is then communicated externally. In some embodiments, the microcontroller  205  takes multiple samples during each cycle of the output signal  204  and averages them to reduce signal noise. The end result is a produced digital value that is more accurate. 
   In some embodiments, some functions (e.g., analog to digital converter) of the microcontroller  205  could be incorporated as a separate circuit. Furthermore, in some embodiments, some circuits (e.g., differential amplifier  270 ) described could also be incorporated in the microcontroller  205 . Where some of the circuits are incorporated in the control system  200  is simply a design choice involving cost, size, accuracy and other factors typical in electrical system design. 
     FIG. 3  describes in more detail the detection circuitry  290  of some embodiments of the invention. The detection circuitry includes, photodiode  170 , operational amplifiers  305   a - 305   d  (collectively operational amplifiers  305 ) and  310   a - 310   c  (collectively operational amplifiers  310 ), gain control circuit  230 , gain control output signal  229 , output signal  239 , analog switch  240 , sampling LED control signal  209 , output signal  249 , “on” hold circuit  250 , “off” hold circuit  260 , output signal  259 , differential amplifier  270 , output signal  269 , analog input signal  204  and several resistors and capacitors, whose values are selected to provide the desired gains for the particular operational amplifiers. 
   Photodiode  170  supplies a small signal to the input of operational amplifier  305   a . Operational amplifiers  305   a  through  305   d  (collectively operational amplifiers  305 ) and their associated resistors and capacitors form amplifier  220  to provide the amplification necessary to detect the signal. Operational amplifier  305   a  converts the current output of photodiode  170  to a voltage. Operational amplifiers  305   b  through  305   d  each provide amplification of the signal using their associated resistors while simultaneously providing some filtering of unwanted noise using their associated capacitors. This use of cascading amplification and filtering provides a much cleaner signal than a single, larger amplifier. 
   The output of amplifier  220  feeds into gain control circuit  230 . Gain control circuit  230  can select between two levels of amplification provided by amplifier  220 . The selection of the level of amplification is done by the microcontroller using gain control output signal  229 . Note that there could be more than two levels of amplification selection if needed. 
   Output signal  239  from gain control circuit  230  goes through operational amplifiers  310   a  through  310   c  (collectively operational amplifiers  310 ). Operational amplifiers  310   a  through  310   c  buffer the signal between gain control circuit  230  and analog switch  240  while simultaneously filtering out noise that may have been introduced by gain control circuit  230 . 
   Analog switch  240  is controlled by sampling LED control signal  209  provided by the microcontroller. Sampling LED control signal  209  also controls the flashing of sampling LED circuit  210  (not shown in this figure). Analog switch  240  will select one of two possible output signals. Output signal  249  will be selected when sampling LED  110  is on, output signal  259  will be selected when sampling LED  110  is off. 
   A resistor and capacitor network form the “on” hold circuit  250  for the signal when sampling LED  110  is on. Similarly, a resistor and capacitor network form the “off” hold circuit  260  for the signal when sampling LED  110  is off. The output signal  259  of the “on” hold circuit  250  is connected to the positive input of differential amplifier  270 . The output signal  269  of the “off” hold circuit  260  is connected to the negative input of differential amplifier  270 . Resulting analog input signal  204  is a signal that is proportional to the fluorescence of the sample. 
   III. Timing of Circuitry 
     FIG. 4  illustrates a timing diagram of the detection circuit  290  in some embodiment of the invention. In an active fluorescent circuit, the sampling LED  110  ideally should not affect the sample. In other words, turning on the sampling LED  110  should not change the steady state fluorescence of the sample. In order to achieve this, the sampling LED  110  should be on for as little time as possible while still allowing the detection circuit enough time to distinguish the fluorescent signal. Thus, the sampling LED control signal  209  has a very short “on” time for the sampling LED  110 . 
   As shown in  FIG. 4 , the sampling LED control signal  209  is on for 50 microseconds and off for 2 milliseconds, in some embodiments. However, other embodiments have on and off times that may vary according to various conditions. As illustrated in  FIG. 4 , the “on” time typically is significantly shorter than the “off” time. 
   In some embodiments, the gain control output signal  239  is an analog signal showing the detection of the fluorescent signal by the photodiode  170  above the ambient light. As shown in  FIG. 4 , the gain control output signal  239  will start to rise as the sampling LED  110  is turned on by the sampling LED control signal  209  and reaches its maximum before the sampling LED  110  is turned off. As further shown in this figure, the gain control output signal  239  returns to the ambient light reading when the sampling LED control signal  209  turns the sampling LED  110  off. 
   In some embodiments, the gain control output signal  239  is the input signal to the analog switch  240 . The analog switch  240  is switched using the same sampling LED control signal  209  that controls the sampling LED  110 . This means that the output signal  249  of analog switch  240  will only see the gain control output signal  239  when the sampling LED  110  is on. Likewise the output signal  259  of the analog switch  240  will only see the gain control output signal  239  when the sampling LED  110  is off. As mentioned above, these signal levels will be held by the hold circuits  250  and  260  respectively. As shown in  FIG. 4 , the “on” hold output signal  268  is a signal that is higher than the “off” hold output signal  269 . Both the hold output signals  269  and  269  are very slow varying signals (i.e., they will not change significantly from cycle to cycle). 
   As previously described, the “on” hold output signal  268  becomes the positive input signal to the differential amplifier  270 , while the “off” hold output signal  269  becomes the negative input signal. The differential amplifier  270  outputs the analog input signal  204 , which is proportional to the difference between the hold output signals  268  and  269 . In some embodiments, the microcontroller  205  will start sampling the analog input signal  204  about 100 microseconds after the sampling LED control signal  209  turns the sampling LED  110  off. In some embodiments, the microcontroller  205  may take multiple samples and averages them to obtain a value for the fluorescent signal. However, the averaging will be completed before the sampling LED control signal  209  turns the sampling LED  110  on for the next cycle. 
   One useful feature of some embodiments is that the sample LED  110  and the detection circuitry can remain synchronized by using the same control signal. In the general use of the fluorometer, the sampling LED  110  sends a signal (the light) into the sample, and the sample&#39;s response to that light is measured. It is useful if the detection circuitry is set up so that it detects when the response is actually happening. So in some embodiments, the design circuitry uses only one signal, namely the sampling LED control signal  209 , to control both the sampling LED  110  and the detection circuitry  290 . With only one signal turning on both the sampling LED  110  and the detection circuitry  290  the whole system remains synchronized, which enhances the accuracy of the measurement. In alternate embodiments which use separate signals control the LED  110  and the analog switch  240 , small differences in timing lead to significant measurement errors. 
   IV. Method for Measuring Photosynthesis Parameters 
     FIG. 5  conceptually illustrates a typical process  500  performed by the microcontroller  205  to gather information on a photosynthetic process. This process is generally performed on a sample of water in which photosynthetic material is potentially present. Often this material would be some form of plant life, or other photosynthetic life, or bits of such life. As shown in this figure, the process begins by waiting (at  510 ) for a sample to “dark adapt”. During this step, the sample is left in the dark for a period of time (anywhere from a few seconds to 10 minutes) so that very little photosynthesis is taking place. This causes the sample to fully utilize any light available for photosynthesis when it becomes available, which also gives a minimum fluorescent signal (“Fo”). 
   In some embodiments, once the sample is dark adapted (at  510 ), the process takes (at  520 ) a measurement or series of measurements of the Fo, using the sampling LED  110  to illuminate the sample, to obtain a minimum fluorescent value. The illumination by the sampling LED  110  causes the sample to fluoresce, the detection circuitry  290  then detects this fluorescence. After obtaining (at  520 ) the minimum fluorescent value, the process turns on (at  530 ) the set of one or more saturating LEDs  130 . The process leaves (at  540 ) the set of LEDs  130  on for some period of time. In some embodiments, the process leaves the set of LEDs  130  on for one second. In other embodiments, the process leaves the one or more saturating LED  130  for a varying amount of time. Some embodiments allow a user to select the period of time for leaving “on” the saturating LED. 
   After leaving the one or more saturating LED(s)  130  for a period of time, the process turns off (at  550 ) the one or more saturating LED(s)  130 . In some embodiments, the process takes (at  560 ) a measurement of a maximum fluorescent signal (Fm), using the sampling LED  110  to illuminate the sample, to obtain a maximum fluorescent value. The illumination by the sampling LED  110  causes the sample to fluoresce, the detection circuitry  290  then detects this fluorescence. This measurement is made as quickly as possible after turning off (at  550 ) the one or more saturating LED  130 . After taking (at  560 ) the Fm, the process continues to take (at  570 ) additional measurements of the fluorescent signal generated by the sample, using the sampling LED  110  to illuminate the sample. The illumination by the sampling LED  110  causes the sample to fluoresce, the detection circuitry  290  then detects this fluorescence. These measurements continue until the fluorescent signal returns to the Fo level, in some embodiments. Some embodiments use these additional measurements to determine other parameters of the photosynthetic process. Once the process measures the Fm, the process determines (at  580 ) a yield for the sample. The yield is defined as the difference between Fm and Fo divided by Fm. The yield is a measurement of the health of the living things in the water, a high yield indicates they are healthy, a low yield indicates they are unhealthy. 
   In some embodiments of the invention, measurements are taken using the sampling LED while the saturating LED is still on at  540 . In such embodiments the photodiode  170  has a high enough dynamic range to distinguish between fluorescence caused by the sampling LED  110  and fluorescence caused by the saturating LED  130 . In some such embodiments, the maximum measurement of the fluorescence taken at  540  is used as the Fm, rather than the value measured at  560 . 
   The invention can be used to determine multiple parameters of the photosynthetic process.  FIGS. 6 through 8  illustrate different protocols that can be used to determine an increasing number of parameters. 
     FIG. 6  illustrates a protocol that determines the yield of the photosynthetic process. This protocol involves dark adapting the sample as described above and taking one fluorescent measurement to measure Fo, using the sampling LED  110  to illuminate the sample, to obtain a minimum fluorescent value. The illumination by the sampling LED  110  causes the sample to fluoresce, the detection circuitry  290  then detects this fluorescence. Then the saturating LED  130  is turned on for some period of time (typically 1 second), and taking a second measurement immediately after saturating LED  130  turns off to measure Fm. Variable fluorescence (Fv) is determined by subtracting Fo from Fm. Yield is determined by dividing Fv by Fm, giving a number between 0 and 1. 
   As shown in the figure, graph  610  of sampling LED  110  shows the “on” or “off” state of the sampling LED  110  (not shown in  FIG. 6 ) versus time. The spikes  615  represent the sampling LED  110  being turned on for a brief time and then turned off for a longer period of time. 
   Graph  630  of saturating LED  130  shows that saturating LED  130  has an “on” period  635  ending just before the sampling LED  110  turns on for the second time. Graph  640  represents the relative fluorescence versus time. Section  645  represents the relative fluorescence while the saturating LED  130  is on. Section  655  represents the relative fluorescence just after the saturating LED  130  has been turned off and the sampling LED  110  has flashed. 
     FIG. 7  illustrates a protocol that measures the response curve of the sample. The protocol starts by duplicating the protocol of  FIG. 6 . However, in this protocol, measurements continue to be made after Fm has been measured. The measurements continue until the sample&#39;s fluorescent output diminishes back to its Fo value. The resulting response curve is another measure of the photosynthetic process. It is the prime measurement used in an instrument to protect natural water supplies against chemical or biological hazards as described by Miguel Rodriguez, Jr. et. al. in the article  Sensors For Rapid Monitoring Of Primary - Source Drinking Water Using Naturally Occurring Photosynthesis  published in the Spring 2002 edition of the journal  Biosensors and Bioelectronics.    
   As shown in the figure, graph  710  of sampling LED  110  shows the “on” or “off” state of the sampling LED  110  (not shown in  FIG. 7 ) versus time. The spikes  615  represent the sampling LED  110  being turned on for a brief time and then turned off for a longer period of time. Note that in  FIG. 7  there are many spikes  615  after the Saturating LED  130  has been turned off. 
   The graph  730  of saturating LED  130  shows that saturating LED  130  has an “on” period  635  ending just before the sampling LED  110  turns on for the second time. Graph  740  represents the Relative Fluorescence versus time. Section  645  represents the relative fluorescence while the saturating LED  130  is on. Section  755  represents the relative fluorescence just after the saturating LED  130  has been turned off and the Sampling LED  110  begins flashing repeatedly. 
     FIG. 8  illustrates another protocol that determines the functional absorption cross-section of PS2 (σ PS2 ). In this protocol saturating LED  130  is pulsed instead of left on continuously. During the period of time when saturating LED  130  is off a measurement is made. This allows a curve to be generated of the fluorescent response of the sample as it is being saturated. Measuring the initial slope of this response gives a measurement of σ PS2 . This method can be refined by varying the pulse duration of the saturating pulses as well as the pulse intensity. 
   By varying the combination of saturating pulse duration and intensity and sampling times, many protocols can be developed to measure additional parameters of the photosynthetic process. Examples of additional protocols can be found in Kolber III with further protocols likely to be developed as knowledge in the field expands. 
   As shown in the figure, graph  810  of sampling LED  110  shows the “on” or “off” state of the sampling LED  110  (not shown in  FIG. 8 ) versus time. The spikes  615  represent the sampling LED  110  being turned on for a brief time and then turned off for a longer period of time. Note that in  FIG. 8  the spikes  615  are closer together than in  FIG. 6 , after the Saturating LED  130  has been turned off. 
   The graph  830  of saturating LED  130  shows that saturating LED  130  has several “on” periods  835  ending just before the sampling LED  110  turns on each time. Graph  840  represents the Relative Fluorescence versus time. Section  845  represents the relative fluorescence while the saturating LED  130  is on and before the sample is saturated. Section  855  represents the relative fluorescence while the saturating LED  130  is on and after the sample is saturated. Section  865  represents the relative fluorescence just after the saturating LED  130  has been turned off and the Sampling LED  110  continues to flash repeatedly. 
   As mentioned previously, in the description of  FIG. 5 , some embodiments use a photodiode  170  that has a high enough dynamic range to distinguish between ambient light, fluorescence caused by saturating LED  130 , and fluorescence caused by sampling LED  110 . These embodiments allow measurements to be made, using the sampling LED, while the saturating LED stays on. In such embodiments the graph  830  would look like the graph  630  from  FIG. 6 . 
   V. Alternative Embodiments 
     FIG. 9  illustrates an alternative embodiment of the invention. As shown in this figure, the sample container is a cuvette  905  that is square. However, in other embodiments, the cuvette  905  can be round. In some embodiments, the cuvette  905  is made of glass or plastic. As further shown in this figure, the photodetector  170  and its associated optical components (e.g., optical components  150 ,  160 , and  190 ) are placed on the bottom of cuvette  905  facing up to detect the emitted light. In some embodiments, two sampling LEDs  110  and their associated optical components (e.g., optical components  120 ,  125 , and  180 ), shine light into the side of the cuvette  905 . In other embodiments, there can be more or fewer than two sampling LEDs  110 . 
     FIG. 9  further shows six saturating LEDs  130  that are placed on the remaining sides of cuvette  905 . In some embodiments, there can be more or fewer than six saturating LEDs  130 . In some embodiments, this configuration of the saturating LEDs  130  provides the advantage of placing several LEDs around the sample while keeping them all orthogonal to the photodetector  170 . 
     FIG. 10  illustrates another alternative embodiment for an application where the sample container is a round flowcell  1005 . In some embodiments, a liquid flows through the round flowcell  1005 . As shown in this figure, the photodetector  170  and its associated optical components (e.g. optical components  150 ,  160 , and  190 ) are placed on a side of the flowcell  1005 . Furthermore, the two sampling LEDs  110  and their associated optical components (e.g., optical components  120 ,  125 , and  180 ) shine light into the side of the flowcell  605  that is orthogonal to the photodetector  170 . In some embodiments, there can be more or fewer than two sampling LEDs  110 .  FIG. 10  further shows four saturating LEDs  130  that are placed opposite to the sampling LEDs  110 . In some embodiments, there can be more or fewer than four saturating LEDs  130 . 
   In some embodiments, using more saturating LEDs  130  may be required if the sample container  105 , cuvette  505 , or flowcell  1005  has a large volume that holds a large sample size, which requires more light to accurately measure the fluorescent response of the sample. 
     FIG. 11  illustrates yet another alternative embodiment for an application where the invention is housed in watertight container  1130 . In some embodiments, the watertight container  1130  is used in submersible applications. As shown in this figure, an optical fiber  1120  is used to carry light from the sampling LED  110  to the ambient liquid. Similarly, an optical fiber  1110  carries the resulting emission light from the ambient liquid to the photodetector  170 . As further shown in this figure, several saturating LEDs  130  (in this illustration  8 ) are placed in a ring around the ambient liquid at the end of optical fibers  1110  and  1120 . In some embodiments, there can be more or fewer than 8 saturating LEDs  130 . 
     FIG. 12  illustrates another alternative embodiment for an application where the invention is housed in watertight container  1130 . This embodiment is different from the embodiment illustrated in  FIG. 11 . As shown in  FIG. 12 , both the sampling LEDs  110  and the saturating LEDs  130  are arranged in the same plane. The detection optics, including an aperture  190 , an emission filter  160 , a lens  150  and photodiode  170 , are separated from the water by a window  1210 . As further shown in this figure, several saturating LEDs  130  (here 6) are placed, along with several sampling LEDs (here 3) in a ring around the ambient liquid outside the window  1210 . In some embodiments, there can be more or fewer than 6 saturating LEDs  130  and/or more or fewer than 3 sampling LEDs  110 . 
   Some embodiments, including some submersible embodiments, use very little power. In some cases the maximum power consumption of the fluorometer may be 10 watts, 5 watts, 2.5 watts, or even 1 watt. Generally, the maximum power consumption occurs when the saturation LED  130  is on. When the saturation LED is off, the power consumption of some embodiments drops below 1 watt. 
   While the invention has been described with reference to numerous specific details, one of ordinary skill in the art will recognize that the invention can be embodied in other specific forms without departing from the spirit of the invention. Thus, one of ordinary skill in the art would understand that the invention is not to be limited by the foregoing illustrative details, but rather is to be defined by the appended claims.

Technology Category: 3