Electronic test standard for fluorescence detectors

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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 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. 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 . 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. 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. 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. 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. 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. 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. 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 (10ˆ -6) than the excitation intensity both optical filters and suitable electronic components can be chosen to produce maximum stability. 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. 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 . 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 &equals;V/Z 3 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. 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 &equals;&agr;I 3 where &agr; is a proportionality constant. The value of &agr; 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. The output voltage V of the operational amplifier 64 may be expressed as V&equals;G ( I 1 Z 1 −I 2 Z 2 ) where G is the gain of the operational amplifier 64 . Since V&equals;I 3 Z 3 , by replacement we can arrive at the expression V&equals;I 2 Z 3 /&agr; and therefore I 2 &equals;( Z 1 /Z 2 ) I 1 &lsqb;1&plus;( Z 3 /&agr;GZ 2 )&rsqb;. 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. 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.