IMPROVED FLUOROMETER ASSEMBLY AND METHOD

Abstract: A fluorometer assembly (60) which is capable of continuously monitoring the performance of its source of excitation energy during the operation therof. A reference photo diode (50), or the equivalent of such a device, is placed along the optical pathway of the excitation energy and continuously senses the output of the source of said energy concurrent with the deflection of a portion of said energy onto a specimen containing the analyte of interest. In the event of deterioration of the source of excitation energy due to aging, or variations in temperature, the reference diode (50) will detect such drift and provide adequate information through a monitoring circuit to compensate for such variation at the time of interpretation of the level of fluorescence from the irradiated specimen. This fluorometer assembly is highly sensitive and provides real-time feedback of fluorometer performance thus making it suitable for use in precision analytical instrumentation.

Description: Improved Fluorometer Assembly and Method BACKGROUND OF THE INVENTION Field of the Invention: This invention is directed to an improved apparatus and to a method. More specifically, this invention concerns itself with an improved fluorometer assembly and a method for continuously monitoring the performance of the source of excitation energy within said assembly during the operation thereof. Description of the Prior Art: The use and advantages inherent- in fluorome- ter . s for analytical instrumentation is well known. One of the more effective environments for use of this tool is referred to as "front surface fluorome- try". Typically, excitation energies are transmitted from a light source within -the assembly, through an appropriate optical band pass filter onto the surface of a specimen, or medium containing the specimen, to be subjected to analysis. The absorption of energy by the specimen results in a secondary emission of light at a lower wavelength and lesser intensity which is proportional to the concentration of the absorbing species within the speci¬ men. The need for precision in instrumentation of this type need not be belabored. Periodic calibration of analytical instruments containing fluoro- meters is routine. In addition to the need for such calibration, due to changes in reagents, the aging of electronic components and temperature variations can also result in drift in performance of the components thereof from their original design and operational specifications. Where such drift occurs, it is impera- tive to immediately recognize such variation and, accordingly compensate for such variation in the evaluation of the fluorescence readings from the specimen which is subjected to fluorometric analysis. For example, if the source of excitation energy gradually deteriorates, or the power supplied to such source fluctuates, the amount of energy striking the specimen may be somewhat less than that programmed into the instrument. When this occurs, the secondary emission from the specimen will not accurately correlate with the concentration of the absorbing species therein and thus comparison of this secondary emission to a standard curve will not accurately reflect the concentration of the emitting species in the specimen. Because such changes 5 generally occur gradually and over prolonged periods of time, or are the result of a non-recurring or intermittant event, it may not be possible to accurately identify when such changes occur and, thus, which of the specific tests results may be effected thereby. J Presently, compensation for drift is overcome in fluorometric instru¬ ments by what is referred to as "ratioing"; a technique similar to that employed in split-beam spectrophotometry. In fluorometers utilizing the ratioing technique, the source of excitation energy is split into two beams: one beam being directed, to a standard (i.e., a stable fluorophore), while the l~ second beam .from the same source of excitation energy being directed ' onto the specimen. Comparison of the secondary emissions from successive excitation of the standard provides a ratio upon which to compensate for drift, thus, enabling meaningful comparison of emissions from the specimen with a standard curve. Instruments utilizing this ratioing technique, of 2 Q necessity, are quite expensive and bulky because of the redundancy built into their optical systems. There is, thus, a continuing need for instrumentation which can perform in an equivalent fashion and yet are comparatively easy to manufacture and maintain. Ideally, such instrumentation should also be simple to operate and capable of recognition of changes in fluorometer -c performance and compensate for such changes and/or variation in fluores¬ cence measurement in real-time. Accordingly, it is the object of this invention to remedy the above as well as related deficiencies in the prior art. 30 More specifically, it is the principle object of this invention to provide a fluorometer assembly capable of continuously monitoring its own perfor¬ mance during the operation thereof. It is another object of this invention to provide a fluorometer assembly 35 capable of splitting the emission from the source of excitation energy into two (2) energy beams of either equal or dissimilar intensity. It is another object of this invention to provide an analytical instrument capable of performance of accurate and rapid clinical assay which incorpo¬ rates the foregoing fluorometer assembly. Still yet, additional objects of this invention include a method for continuously monitoring the performance of a fluorometer during the opera- tion thereof; and, for the compensation for such variation in fluorometer performance at the time of interpretation of the level of fluorescence from the irradiated specimen. SUMMARY OF THE INVENTION The above and related objects are achieved by providing a fluorometer assembly comprising a source of excitation energy and means for monitoring the output of the source of excitation energy. Such monitoring means are to be disposed along the optical pathway of the source of excitation energy. Between the monitoring means and the source of excitation energy along this optical pathway is located a reflecting means, typically a dichroic mirror - preferably oriented at -.5 degrees to the direction of propagation - which is capable of deflecting at least a portion of the excitation energy to a specimen containing the analyte of interest while at the same time permit- ting the uninterrupted transmission of at least a portion of said excitation energy to the monitoring means. In a preferred embodiment of this invention, the fluorescence emission from the fluorophore contained in the specimen will also be transmitted through the dichroic mirror to a means for detection of the level of such emission (typically a photomultiplier tube). In addition to the foregoing elements, such assembly can also include one or more lenses to further refine the focusing of the various beams of excitation energy and fluorescence emissions; and filters (i.e., band pass, infrared absorption, etc.) to screen out unwanted wavelengths of light. -* BRIEF DESCRIPTION OF THE DRAWINGS 5 Fig. 1 is a perspective view of an analytical instrument designed for performing batch processing of patient samples by fluorometric analysis. Fig. 2 is a diagramatic representation of the fluorometer assembly of the 10 type incorporated within the instrument illustrated in Fig. 1. DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS i c The automated batch analyzer illustrated in Fig. 1 utilizes a unique sel -stacking reagent tab which is stored within the analyzer at tab load station 2. In operation of the analyzer, a single reagent tab (not shown) is peeled from the stack stored within the load station by a pick-off finger (not shown) of the carousel assembly. Th carousel assembly contains two (2 ) r,r, separate and independently rotatable sections which move in registration with one another through the various processing stations of the analyzer. Initially, a reagent tab is transferred from the load station onto a rotating platform of the carousel which precisely orients it relative to each of the three (3) fluid dispensing stations 8, 12 and 16 and fluorometer assembly 60 __ which are positioned at pre-deter mined locations along its circular pathway. The operation of this analyzer can be tailored to the particular type of test that is to be performed and thus may vary from one analytical protocol to another; and, the particular demands of the chemistries which are unique to such protocol. The following description of the operation of this analyzer is made in reference to an enzyme immunoassay (EIA) which is conducted 30 totally within the confines of the reagent tab. At the first such station, a series of cams position the syringe mechanism of pump assembly 8 over sample cup 6 containing a patient specimen. A microprocessor within the instrument activates the syringe 35 mechanisms of the pump through a series of stepping motors, thus resulting in the pump first aspirating and then dispensing an aliquot of patient specimen from the sample cup onto the reagent tab. The patient sample is precisely applied to the center of the reaction zone of the reagent tab. The distance OMPI of the dispensing tip and rate of application of patient sample are precisely controlled by the analyzer logic. After application of the patient sample to the reagent tab, the carousel transfers the reagent tab to the next station within the instrument. The rate of transfer of the tab from one station to the other is carefully programmed to allow adequate time for incubation of the sample with the reagents of the reagent tab. The incubation period between dispensing of sample onto the reagent tab and the. next fluid dispensing station is critical in order to allow for sufficient binding of the analyte of interest from the patient sample to an antibody which is immobilized within the reagent tab. The ambient environment within the analyzer is also carefully controlled in order to optimize this interaction of patient sample and reagent in the tab. Subsequent to dispensing of patient sample to the tab, the dispensing tip of the sample pump arm is repositioned and flushed of residual patient sample with water from a reservoir located at wash station 10. The reagent tab containing the patient sample is precisely positioned under the next aperture in the carousel housing (partially obscured by boom of sample dispensing pump). At this next fluid dispensing station 12, the dispensing tip of the pump aspirates a precise quantity of antigen-enzyme conjugate from reservoir 1 and delivers it to the reagent tab through the aperture in the carousel housing. The constraints built into the tab transport mechanism of the carousel assembly precisely position the reagent tab relative to this station so as to enable application of the conjugate to the center of the reagent tab. The manner of application of conjugate by the conjugate dispensing station is controlled in a similar fashion as was the case in the sample dispensing station. The quantity of conjugate, and its rate of application and distance of the dispensing tip of the syringe from the reagent tab are also precisely controlled in order to insure essentially uniform flow of the conjugate from the dispensing tip into the reagent tab. τ he reagent tab is then positioned under the aperture in the carousel housing at the substrate dispensing station 16 while a series of cams positions the syringe mechanism of the pump 18 over reservoir 20 containing a substrate fluid and a microprocessor activates the syringe through a series of stepping motors to aspirate and then dispense the required aliquot of this 5 fluid onto the reagent tab in a manner similar to that performed previously at the sample and conjugate dispensing stations. The amount of substrate dispensed onto the reagent tab is sufficient to effect radial elution of unbound materials out of the anticipated field of view of the fluorometer at the center of the reagent tab. After a brief incubation period, the substrate 10 interacts with the enzyme portion of the conjugate molecule bound at the center of the tab releasing a fluorophore. The reagent tab, containing the various solutions which have been applied to it at the three (3) immediately preceding dispensing stations, is 5 " now transferred, in relatively rapid succession, to a fluorometer read station of the type diagmatically illustrated in Fig. 2. The field of view of the fluorometer is controlled by aperature 0 and thusly confined to but a small portion of the center of the reagent tab which coincides with the point of fluid application. As illustrated in Fig. 2, reagent tab 6 is illuminated with 20 excitation energy from light source 42. The excitation energy is directed to dichroic mirror 4 which reflects a substantial portion of said energy onto that portion of the reagent tab which has not been masked from such energy by aperture 40. The excitation energy striking the tab causes the fluorophore In the tab to emit fluorescence radiation, a fraction of which is directed 25 toward and through the dichroic mirror and optical filter band pass 56 (that is specific for the fluorescnece wavelength of interest) onto fluorescence sensing means 48, such as a photomultiplier tube. The level of fluorescence is monitored over a finite period in order to construct a curve, the slope of which being indicative of the concentration of analyte in the patient sample. , n Unlike the conventional fluorometric instruments which utilize the ratioing technique for detection of drift in the performance of the source of excitation energy, the assembly illustrated in Fig. 2 continuously monitors the output of said source by placement of a monitoring means 50, such as a reference photo diode, along the optical pathway of the excitation energy. -,. Representative of such a photo diode would be a silicon solid state diode, operated in the photovoltaic mode, which is extremely stable and whose current is proportional to incident radiation. The dichroic mirror is inten¬ tionally designed so as to permit uninterrupted transmission of a pre- determined proportion of excitation energy to the monitoring means. One of the principle functions of this dichroic mirror is to split the excitation energy into any usable ratio. In practice, transmission of from 2 to 5% and reflection of 35 to 98% of the excitation energy provides an acceptable and workable ratio; however, such ratio could in fact be the inverse and still provide an operative system (depending of course upon the intensity of the fluorescence emission from the patient sample and the sensitivity of the means for monitoring such emission.) Any deviation from design specifica¬ tions in the intensity of energy transmitted through the dichroic mirror to the monitoring means is relayed to a microprocessor 52 in the instrument which notes such variation. The microprocessor can automatically compensate for such difference in its comparison of the fluorescence emitted from the irradiated portion of the reagent tab to a standard curve which is stored within the microprocessor. In the particular embodiment illustrated in Fig. 2, a series of filters, 54, 56 and 58, are also positioned between the source of excitation energy and •the various detectors (reference photo diodes and photomultiplier tubes). In addition to such filters, lense assemblies (not shown) may also be positioned in a similar fashion to further refine and focus the various beams of electromagnetic radiation before they impinge upon the element at which each is targeted. In one of the preferred embodiments of this invention, the fluorometer assembly can be contained within a monolithic block which would not only serve to maintain precise positioning of components, but also render the components of such assembly less sensitive to temperature changes. The foregoing description has been provided to illustrate but one of the possible applications of the fluorometer assembly of this invention and not to delineate its scope which is set forth in the following claims. CtvIH

Claims: WHAT IS CLAIMED IS: 1. A fluorometer assembly for use in fluorometric analysis of a specimen containing an analyte of interest, said analysis being based upon either direct or indirect measurement of said analyte from the level of fluorescence associated with such measurement, the assembly compris¬ ing: (a) a source of excitation energy; (b) means for continuously monitoring the emission from the source of excitation energy disposed along the optical pathway of the source of said energy; and (c) reflecting means positioned along the optical . pathway of • the excitation energy between the source thereof and said monitoring means, said reflecting means being adapted to reflect a predetermined portion of excitation energy from said source to a specimen containing an analyte of interest and to allow for the uninterrupted transmission of a predetermined portion of excitation energy from said source through said reflecting means to said monitoring means. 2. The fluorometer assembly of Claim 1, comprising a means for monitor¬ ing fluorescence from the specimen containing the analyte of interest. 3. The fluorometer assembly of Claim 2, wherein the reflecting means is positioned between said specimen and said fluorescence monitoring means along the optical pathway of the fluorescence emission from the specimen, the reflecting means being further adapted to allow for the essentially uninterrupted transmission of a substantial portion of said fluorescence emission from said specimen through said reflecting means to said fluorescence monitoring means. 4. The fluorometer assembly of Claim 1, wherein the reflecting means is a dichroic mirror which is transmissive of at least about 2% of the excitation energy. 5. The fluorometer assembly of Claim 1, comprising a means for defining the field of view of said assembly relative to the specimen. 6. The fluorometer assembly of Claim 1, comprising means for focusing the excitation energy. 7. The fluorometer assembly of Claim 2, comprising means for focusing the fluorescence associated with measurement of analyte onto said fluorescence monitoring means. 8. The fluorometer assembly of Claim 7 in which the means for monitoring excitation energy is a solid State silicon photo diode. 9. A method for real-time correction of errors in fluorescence measure¬ ments in analytical instrumentation which result from characteristic short term fluctuations and/or long term drift in fluorometer excitation energy, said method comprising: (a) splitting a beam of excitation energy of a fluorometer of said instrumentation into at least two (2) useful components, one such component being directed to a specimen containing the analyte of interest and another of such component being directed to a sensing means for continuously monitoring the level of excitation energy during operation of said fluorometer; (b) relaying the response of said sensing means to such excitation energy to a microprocessor; (c) comparing the response of said means to said excitation with predetermined parameters stored in the memory of the micropro¬ cessor; and, ( d) adjusting for any deviation in response from predetermined para¬ meter in interpreting fluorescence measurement.

Inventors: HELLER ZINDEL HERBERT, HELLER, ZINDEL, HERBERT
Applicants: AMERICAN HOSPITAL SUPPLY CORP, AMERICAN HOSPITAL SUPPLY CORPORATION
Application year: 1983
Main classification: G01N21
IPC codes: G01N21