Abstract:
The present invention relates to a self-contained optical repeater that detects light of a first frequency (color) and emits light at a different frequency (color) with intensity related to the incident light flux of the detected light of the first frequency. The emitted light of the second frequency (i.e., the excitation light) is used to fluoresce an optical sample to emit the detected light of the second frequency (i.e., the fluoresced light). The frequency (and energy) of the excitation light greater than the frequency (and energy) of the fluoresced light. The excitation light is filtered and detected by a photodiode. Output of the excitation light is electronically controlled to be a predetermined fraction of the incident fluorescent illumination as filtered and presented to the electronic control circuit in a geometry that mimics a specific fluorescent chemistry. It is important to control the output of the excitation light source to compensate for variations of the light source output with variations in external conditions, such as temperature, to maintain a truly constant ratio between the excitation and fluoresced light intensities.

Description:
TECHNICAL FIELD OF THE INVENTION  
         [0001]    The present invention relates generally to fluorimetry and, more particularly, to a novel method and apparatus for using an electronic calibration standard to calibrate a fluorimeter without the use of a physical fluorescence standard.  
         BACKGROUND OF THE INVENTION  
         [0002]    Fluorimetry is an important quick and nondestructive analytical chemistry technique. Fluorimetry is used to acquire both qualitative and quantitative data, and is of great interest for use in clinical chemistry and medical diagnostics as a means for measuring unknowns such as the pH and partial pressure of blood gasses and blood analytes.  
           [0003]    In general, fluorometric analysis involves shining an energetic light onto a sample and stimulating the immediate re-emission or fluorescence of light of a particular frequency from the sample. The frequency of the light so fluoresced is characteristic of the particular sample component fluorescing. The frequency of the light shined onto the sample is usually chosen to be slightly higher than that of the frequency of the light characteristically fluoresced by the sample component desired to be measured. In other words, the fluoresced light has an energy less than or equal to that of the light source, since conservation of energy and the quantum nature of light dictate that, for single photon processes, the fluoresced photons cannot be more energetic than the excitation photons absorbed to produce the fluoresced photons.  
           [0004]    Fluorimeters are currently calibrated by fluorescing stable materials having well-known fluorescent wavelengths and well-characterized fluorescent intensities as calibration standards. For a homogeneous sample excited by a light source having a given frequency and intensity, the intensity of the fluoresced light is proportional to the quantity of the fluorescent material. So long as the calibration standard is a suitable simulacrum of the sample to be investigated, the requirement for stability of the light source and optical detection system on the fluorimeter is mitigated by the use of a suitable calibrator in conjunction with the measurement. One important feature of this calibrator is to return to the instrument “fluoresced” photons of the appropriate color and at an intensity substantially proportional to the fluorescence excitation.  
           [0005]    Special fluorimeters measure the lifetime of the fluorescent state using pulse and/or phase sensitive techniques. Although these measurements are not directly sensitive to the magnitude of the fluorescent signal, some degree of regularization of signal amplitude is often useful and rudimentary calibration required.  
           [0006]    The currently available fluorescent calibration standard materials suffer from a number of serious drawbacks contributing to measurement errors, but have the overarching advantage of being the only options available for calibrating a fluorimeter. Examples of sources of error afflicting fluorescent standards include the relative rarity of fluorescing materials, the instability of most fluorescing materials under ambient environmental conditions, the inability to stabilize organic fluorescent materials through glass encapsulation, variations in fluorescent intensity between different specimens of the same fluorescent material (intrasample variation) and geometrical differences between the source and the detector from calibration to calibration arising due to variances in sample placement. Therefore, a need has arisen for a fluorescence calibration standard with reduced geometric and intrasample variations and having stable fluorescence properties over time and environmental conditions. The present invention addresses this need.  
         SUMMARY OF THE INVENTION  
         [0007]    The present invention relates to an electrical device for comparing the intensity of the light from a fluorescence excitation source in a fluorimetry instrument to the light emitted from an emulation light source that emulates the light that is otherwise resultingly fluoresced from an optical sample. The electrical device also controls the output of the excitation source to maintain a substantially constant relationship between the intensity of the excitation source and the intensity of the emulation source, and thereby the fluoresced light. The device includes a light source for emulating the fluorescence emission, a first photodetector for measuring the intensity of the excitation light source, a second photodetector for measuring the intensity of the emulated fluorescence, and an electronic circuit for comparing the intensities of the light from the excitation source and the emulation source, and for controlling the intensity of the emulation source to maintain a constant, predetermined ratio between the two that may be used by the instrument for calibration purposes.  
           [0008]    One form of the present invention relates to an electronic fluorescence standard, including a window for receiving light from a fluorescence excitation light source in a fluorimetry instrument, a first photodetector, a fluorescence emulation light source, and a first light pipe extending from the excitation light source to the emulation source and to the first photodetector, a second photodetector, a second light pipe extending from the emulation light source to the second photodetector, and an electronic controller operationally connected to the emulation light source, the first photodetector and the second photodetector, wherein the second light pipe is adapted to direct light from the emulation light source to the second photodetector, wherein the first light pipe is adapted to direct light from the excitation light source to the first photodetector, wherein the first and second photodetectors are adapted to respectively send a first and a second output current to the electronic controller proportional to the light received by the respective photodetector, and wherein the electronic controller is adapted to compare the first and the second output currents and adjust the light output of the emulation light source to achieve a predetermined relationship between the first and the second output currents.  
           [0009]    One object of the present invention is to provide an improved apparatus for calibrating a fluorimeter. Related objects and advantages of the present invention will be apparent from the following description.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]    [0010]FIG. 1 is a schematic illustration of the fluorescence meter calibration device of a first embodiment of the present invention.  
         [0011]    [0011]FIG. 2 is a schematic illustration of an electronic control circuit in the electronic controller of FIG. 1.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0012]    For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.  
         [0013]    The present invention relates to a method and apparatus for calibrating a fluorimeter. FIG. 1 illustrates one embodiment of the present invention, an electronic fluorimeter calibration device  20  positionable in a fluorimeter. The calibration device  20  is capable of detecting light of a first predetermined color and emitting light of a second, different predetermined color with intensity related to the detected light flux. The calibration device  20  includes a fluorescense emulation light source  22  positioned to shine through an optically transparent window  24  in the calibration device  20  and onto a component of a fluorimetry instrument, such as the fluorescence excitation light source  26  or a calibration photodetecotor  28 . The emulation light source  22  is preferably a light-emitting diode (LED), but may be of any convenient design capable of providing light of sufficient energy and frequency to emulate the light characteristic of a desired fluorescence target material. The fluorescence emulation light source  22  is preferably positioned to shine through the window  24  through a light pipe  30  positioned between the emulation light source  22  and the window  24 . The light pipe  30  is preferably able to direct light from the emulation source  22  through the window  24  through the process of internal reflection. However, any optical device or system capable of efficiently directing light from the emulation light source  22  to the window  24  may be chosen. More preferably, an opaque optical shield  32  is formed around the window  24  formed therein and is positioned such that light from the emulation light source  22  is guided through the light pipe  30  and through the window  24  to shine onto the calibration photodetector  28 . The opaque optical shield  32  thereby substantially prevents extraneous light from shining through the window in either direction and contributing to measurement error. The light pipe  30  may also preferably be configured to guide light from the optical sample excitation source  26  to a first photodetector  34 . As illustrated in FIG. 1, the light pipe  30  is preferably generally Y-shaped, with a first leg  36  extending from the emulation light source  22  to the window  24  and a second leg  38  extending from the window  24  to the first photodetector  34 . The light pipe  30  may, however, have any convenient shape functional to guide light from the emulation light source  22  to the window  24  and from the window  24  to the first photodetector  34 .  
         [0014]    A second photodetector  40  is positioned to receive light from the emulation light source  22 . Preferably, the light from the emulation light source  22  is guided to the second photodetector  40  by a second light pipe  42 , although the second photodetector  40  may be positioned to receive light directly from the emulation light source. As with regards to the first light pipe  30 , the second light pipe  42  preferably directs light from the emulation light source  22  to the second photodetector  40  through total internal reflection, but may alternately do so through any other convenient light directing process. More preferably, the emulation light source  22  is shielded from directly shining onto the first and/or the second photodetector  34 ,  40 , such as by the placement of an opaque shield  44  therebetween.  
         [0015]    The first and second photodetectors  34 ,  40  are electrically connected to an electronic controller  46 . The electronic controller  46  is also electrically connected to the emulation light source  22 . The electronic controller  46  includes circuitry adapted to compare the inputs from the two photodetectors  34 ,  40  and to change the output of the emulation light source  22  in order to maintain a preselected relationship between the outputs of the two photodetectors  34 ,  40 , and to therefore allow the emulation light source  22  to maintain the frequency and intensity of the fluorescent material it is desired to emulate.  
         [0016]    The calibration device  20  is preferably configured as a cartridge, compatible to be plugged into a fluorimeter for calibration as required. However, the calibration device  20  may also be configured as a built-in feature of a fluorimeter. The surface of one or more of the optical elements (i.e., the light pipe(s)  30 ,  42 , the filter  52 , the window  24 ) may be optically textured such that the light from the emulation source  22  more closely resembles the light from the true fluorescent source it emulates. For example, if the emulated fluorescence source is characterized by diffuse emission, a diffuser or diffusing coating may be applied to one or more of the optical elements such that the calibration device  20  more closely emulates the character of the light emitted from the emulated fluorescence source.  
         [0017]    In operation, the calibration device  20  functions to simulate the scattering geometry and fluorescence of the chemistries associated with a particular fluorescence meter system. Light from the excitation light source  26  of the fluorimeter is directed to the first photodetector  34 . Light from the fluorescence emulation light source is directed to the second photodetector  40  and is sampled thereby. The first and second photodetectors  34 ,  40  each send a signal to the electronic controller  46  proportional to the intensity of the incident light from the respective light sources  26 ,  22 . The second photodetector  40  is chosen to have its peak frequency sensitivity range coincide with the peak frequency of the light source  22 , with photodetectors  40  having different peak frequencies paired with emulation light sources  22  of different peak frequencies to calibrate the fluorimeter for different fluorescent materials. In other words, since a given fluorescent material emits fluorescent light having a characteristic peak frequency, an emulation light source  22 /second photodetector  40  pair is chosen to respectively emit and detect light of a frequency matched to that of the fluorescent material for which the fluorimeter is desired to be calibrated. Likewise, the first photodetector  34  is also preferably chosen to have its peak frequency sensitivity range coincident with the peak excition frequency.of the fluorescent materials.  
         [0018]    The electronic controller  46  automatically converts the currents from the photodetectors  34 ,  40  to voltages and compares the voltages. The electronic controller  46  then automatically generates an amplified response voltage, which is then converted to a current to drive the emulation light source  22 . The circuit automatically tries to eliminate or substantially minimize the difference in current (or transimpedance amplified voltage outputs) between the signals from the photodetectors  34 ,  40 . This is accomplished by varying the response voltage, and therefore the current driving the emulation light source  22 , such that the output of the emulation light source  22  is varied until the signals from the two photodetectros  34 ,  40  are substantially identical.  
         [0019]    The calibration device  20  is therefore a self-contained optical repeater that detects light of a predetermined frequency or color, and emits light of a lower frequency (different, less energetic color) with intensity governed to satisfy a predetermined intensity relationship between the detected light of the first frequency and the emitted light of the second frequency.  
         [0020]    The calibration device  20  preferably includes a filter  50  between the light from the excitation source  26  and the first photodetector  34 . A emulation source filter  52  is likewise preferably positioned between the emulation source  22  and the second photodetector  40 . The efficiency of the filters  50 ,  52  for reducing the intensity of the light shining therethrough and onto a respective photodetector  34 ,  40  determines the effective intensity of the light passing therethrough to shine on a respective photodetector  34 ,  40  and therefore the intensity of the current generated by the respective photodetector  34 ,  40  to be sent to the electronic controller  46 . By properly selecting the efficiency value of the filters  50 ,  52  the relative intensities of the lights generated by the excitation light source  26  and the emulation source  22  may be controlled. In principle this control could be accomplished via the electronics, but because the emulation intensity may be much smaller (10^ -6) than the excitation intensity both optical filters and suitable electronic components can be chosen to produce maximum stability.  
         [0021]    [0021]FIG. 2 illustrates one example of an electronic controller  46  circuit design adapted to compare the inputs from the two photodetectors  34 ,  40  and to change the output of the emulation light source  22  in greater detail. There are many electronic circuit designs capable performing the servometric function, this approach is illustrative of one straightforward method. A first transimpedance amplifier  56  is connected to the first photodetector  34 , such that the output current from the first photodetector  34  is received as by the input  58  by the first transimpedance amplifier  56 . Likewise, a second transimpedance amplifier  60  is connected to receive the output current from the second photodetector  40  through the second transimpedance amplifier input  62 . An operational amplifier  64  is provided having a non-inverting input  65  connected to the output  66  of the first transimpedance amplifier  56  and an inverting input  67  connected to the output  68  of the second transimpedance amplifier  60 . The output  70  of the operational amplifier is electrically connected to the input  72  of a transconductance amplifier  74 . The output  76  of the transconductance amplifier  74  is electrically connected to the anode  78  of a light-emitting diode  22 , the cathode  82  of which is connected to a ground potential.  
         [0022]    In operation, a first current I 1  is generated by light incident upon the first photodetector  34 . The first current I 1  is proportional to the intensity of the light on the first photodetector  34 . Likewise, a second current I 2  is generated by and proportional to light incident upon the second photodetector  40 . The current I 1  from the first photodetector is input into the first transimpedance amplifier  56  and transformed into a voltage output having a voltage equivalent to I 1 Z 1 , where Z 1  is the transimpedance value of the first transimpedance amplifier  56 . Similarly, the second transimpedance amplifier  60  (having a transimpedance value of Z 2 ) outputs a voltage of I 2 Z 2  in response to a current input I 2 .  
         [0023]    The operational amplifier  64  has a gain of G and receives the voltage I 1 Z 1  input at the non-inverting terminal  65  and the voltage I 2 Z 2  at the inverting terminal  67 , and outputs a voltage V in response. The voltage V is the voltage input to the transconductance amplifier  74 , which outputs a current I 3  according to the equation  
         
       I 
       3 
       =V/Z 
       3  
     
         [0024]    where Z 3  is the transconductance value of the transconductance amplifier  74 . The current I 3  is then output to the anode  78  of the light emitting diode  22 , where it is used to drive the photonic emission of the light-emitting diode  22 . In other words, the intensity of the light emitted from the light-emitting diode  22  is proportional to the current I 3  flowing thereinto.  
         [0025]    Since the current I 2  flowing from the second photodetector  40  is proportional to the light shining onto the second photodetector  40  from the light-emitting diode  22 , and the light emitted from the light-emiting diode  22  is proportional to the current I 3  flowing therethrough, the current I 2  is proportional to the I 3 . Therefore,  
         
       I 
       2 
       =αI 
       3  
     
         [0026]    where α is a proportionality constant. The value of α is a function of the efficiency of the light-emitting diode  22 , the efficiency of the transmission of the light from the light-emitting diode  22  to the second photodetector  40 , and of the efficiency of the second photodetector  40  in converting light energy to current.  
         [0027]    The output voltage V of the operational amplifier  64  may be expressed as  
           V=G ( I   1   Z   1   −I   2   Z   2 )  
         [0028]    where G is the gain of the operational amplifier  64 . Since V=I 3 Z 3 , by replacement we can arrive at the expression  
           V=I   2   Z   3 /α 
         [0029]    and therefore  
           I   2 =( Z   1   /Z   2 ) I   1 [1+( Z   3   /αGZ   2 )].  
         [0030]    So long as the second bracket term is small, the fluoresced light intensity will be substantially proportional to the excitation light intensity, depending only on the constancy of the transimpedance ratio. This criterion is easily met in practice.  
         [0031]    While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are to be desired to be protected.