Abstract:
A device for photometric measurement of several samples ( 12 ) that are exposed to radiation from a light source ( 14 ) associated therewith. The light modified by the samples ( 12 ) is intercepted by an optical device ( 19 ) and is guided as a sum of all the light radiated from all samples ( 12 ) to at least one sensor ( 27 ) for measuring the intensity and evaluation thereof in an evaluation device ( 32 ) arranged downstream. The light sources ( 14 ) are controlled individually by a control device ( 31 ) and the evaluation device ( 32 ) and the control device ( 31 ) are controlled such that the evaluation device ( 32 ) separates the light from each sample ( 12 ) from the light of the other samples. The control device ( 31 ) modulates the light source ( 14 ) with various signals, which are pre-assigned to the evaluation device ( 32 ) and used to determine the light signals of the individual samples ( 12 ) from the sum signal received by the sensor ( 27 ).

Description:
BACKGROUND OF THE INVENTION 
   The present invention relates to an apparatus for photometrically testing several specimens ( 12 ) that are each irradiated by an associated light source. An apparatus of this type is used to photometrically test several specimens, for instance on-line on a thermocycler, in order to monitor the progress of PCR procedures. However, such an apparatus is also used to test a plurality of microarray spots on glass slides, or as well wherever a plurality of specimens must be tested photometrically by means of fluorescence, absorption, scattering phenomena, and the like. When testing fluorescence specimens, an appropriate fluorescence indicator is added as a rule. 
   Such an apparatus, however, incurs the drawback of entailing a plurality of light paths, which, in turn, require a complex design. When only one light path is used between a light source and a light sensor, then the specimens must be moved sequentially into the light path and substantial mechanisms are entailed. It is furthermore known to move the light, such as a reflected light, over stationary specimens. Unfortunately, this design incurs the same drawbacks as above. 
   On the other hand, to simultaneously irradiate all specimens simultaneously by the same light source and to view them individually using several sensors would be exceedingly costly. This is due, in part, to the fact that the sensors are much more expensive than light sources. 
   Accordingly, the patent documents EP 0902271 A2 and WO 01/35079 A1 propose individually irradiating the specimens using associated light sources and to use only one light sensor to determine and analyze the light from all specimens, namely the summed signal. 
   This solution, however, raises the problem of identifying the light intensities of the particular specimens from the summed signal picked up by the sensor. The state of the art disclosed by both of the above patent documents solves this problem in that the different light sources are operative only individually, so that only the light from a given specimens will fall on the sensor which only receives a single specimen&#39;s light that, provided there be appropriate synchronization, can be allocated to the particular specimen. 
   However such apparatus suffers from drawbacks because, assuming there are n specimens, each specimen may be illuminated at most only 1/n of the available measuring time. The values related to the particular specimens are available only within short time intervals. Moreover, the measured signal cannot be integrated over a substantial length of time, illustratively when the signal is weak, to still attain in this manner a good signal to noise ratio. If the signal fluctuates, or when ascertaining the progress of a reaction in the specimens, it will be impossible to monitor the signal as a function of time. 
   SUMMARY OF THE INVENTION 
   Therefore, it is an object of the present invention to design an apparatus of the above kind such that the specimens can be measured over comparatively long time intervals. 
   In accordance with the invention, all specimens are constantly illuminated and analyzed. The illumination is carried out using differently modulated signals, the summed signal transmitted by the sensor being resolvable into the individual signals when knowing the different modulations of the signals provided the different signals do differ sufficiently. The summed light from the specimens may be analyzed by one or more sensors that analyze, for instance, different effects appearing in the light. The light sources are individually driven by a control unit. Alternatively, the control unit may be in the form of several control units individually associated with the light sources. Individual light source control allows driving the light sources individually or in groups, illustratively one group of identical specimens being irradiated with the same signal modulation. The signal modulation must be known to the analyzer to allow the analyzer to analyze the individual signals. Illustratively, such knowledge may be implemented by permanently programming into the analyzer the modulations being used or by transmitting the modulations from the control unit. Under particular default conditions, the analyzer also may itself retrieve the modulations. A substantial advantage is offered by the feasibility of permanently acquiring the signals from all specimens, whereby these signals may, for instance, be integrated over long time intervals or be monitored regarding their changes with time. The apparatus of the invention is applicable to all optical measurement procedures concerning specimen analysis such as measuring fluorescence, transmission, light scattering, and the like. The apparatus may be used for on-line specimen determination on thermocyclers (for PCR), chip readers, MTP readers, spot readers, and other multi-analytical test means. 
   In further accordance with the present invention, the signals are square. This creates advantages in circuitry and also regarding signal analysis. Particularly, square signals offer the advantage of digitized signal generation. 
   The signals must appropriately differ in their modulations. Illustratively, they may differ with respect to phase. However, in accordance with the present invention, the signal modulations differ in their frequencies. The individual signals may be separated in the analyzer using, for instance, appropriate frequency filters. The circuits required are comparatively simple. 
   Alternatively, and in further accordance with the present invention, the signals differ in their code sequence. Using appropriate decoding algorithms, the individual signals may be retrieved from the summed signal. Advantageously, the signals are mutually orthogonal to allow clean signal separation even for a plurality of such signals, such as when several hundred specimens must be analyzed. 
   In further accordance with the present invention, the control unit generates the frequencies according to a specific algorithm from a single mother clock timing and the analyzer, using the algorithm, generates the frequencies required for analysis from the same mother clock timing procedure. In this manner many different frequencies may be generated in highly accurate and precise manner, both to drive the light sources and to analyze the summed signal. 
   Frequency-discriminated signals may be retrieved by individually multiplying the summed signal with each particular signal frequency and by subsequent lowpass filtering. However, in further accordance with the present invention, the analyzer retrieves the individual signals out of the summed signal by Fourier transforms and by determining the amplitudes of the individual signal frequencies. Signal frequency analysis of the individual specimens from the summed signal by Fourier transformation is highly accurate and precise and its circuitry is easily implemented. 
   The suitable light sources may be all those fit for photometry that may selected depending on the requirements on light, for instance for fluorescence purposes—or depending on the requirements set on the light path—for instance sharp focusing on one of several specimens—or regarding light intensity or also the rate of modulation. Accordingly, both incandescent bulbs and laser, flash and other light sources are applicable. Advantageously, however, the light sources are light emitting diodes or LEDs. LEDs or laser diodes are economically available while offering appropriate light quality and are characterized by being easily integrated and mounted, for instance as an array on a printed circuit board. 
   Preferably, light sources are selected such that the light radiated by them is able to excite selected fluorescence colors. 
   In further accordance with the present invention, a filter to eliminate interfering light is configured in the downstream direction of the radiation of the light sources. A short wavelength filter is used in fluorescence measurements to filter out light of long-wave lengths, in particular in the region of the fluorescence light. Bandpass filters also are suitable. 
   In accordance with another aspect of the invention, light from the specimens to the sensor is deflected by a dichroic beam splitter transmitting the light from the light sources. The dichroic beam splitters further improve the separation between the short wavelength light of illumination and the long wavelength light of fluorescence. 
   In accordance with another aspect of the invention, the optical device includes comprises several optical-guides/fibers of which the light entry surfaces are mutually spaced and parallel and of which the light exit surfaces are adjacent and parallel. The collimator required to collect the light radiated from the specimens and transmitting it to the light sensor may be improved for instance with respect to the conventional lens element collimator, especially as regards the parallelism of the light incident on the sensor. This high parallelism is especially advantageous when the sensor would be susceptible at its input to deviations from the light&#39;s parallelism, for instance when being fitted at the input with an interference filter. 
   In further accordance with the present invention, a filter eliminating interfering light is situated in front of the sensor. As regards fluorescence measurements, the filter is a long wavelength filter eliminating residual short wavelength portions of the light illustratively arising as scattered light from the light sources. Bandpass filters are also appropriate for these purposes. 
   Further, the specimens may be configured in wells of a thermocycler and may be tested photometrically on a thermocycler for the purpose of optimizing a PCR procedure carried out on the thermocycler. 

   
     BRIEF DESCRIPTION OF THE DRAWING 
     These and further features of the invention will be apparent with reference to the following description and single drawing FIGURE, which illustrates the preferred embodiment of the inventive apparatus. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
   The apparatus  10  comprises a schematic, conventional thermocycler  11  with wells  12 . Reaction vials (not shown) are in place in the wells  12 . Each vial contains one PCR batch with one or more fluorescence indicators. 
   A covered housing  13  fitted with an illumination unit of several LEDs  14  is set on the thermocycler  11 . One LED  14  is allocated to each well  12 . Preferably, the LEDs  14  are configured as an array. Each LED  14  points in a direction such that it will irradiate only one associated well  12  and, if possible at all, not the adjacent wells. The LEDs  14  may, in particular, be laser diodes. 
   An illustrative light path is denoted by  15 ,  15 ′. The light  15  is radiated from the LED  14  and first passes through an optional, but preferred, short wavelength filter  16  by means of which long wavelength components are filtered out. Then the light  15  passes through a beam splitter  17 , which, in this instance, preferably shall be wholly transmitting. 
   The light  15  radiated from the LED  14  will excite a fluorescence indicator contained in a PCR batch in the well  12 . The indicator, in turn, emits a fluorescence signal  15 ′. The beam splitter  17  is designed such that the fluorescence signal  15 ′ is reflected laterally. 
   A dichroic mirror is preferably used as the beam splitter  17  and will transmit the excitation light while reflecting the emitted, longer wavelength fluorescence signal. 
   The reflected fluorescence signal  15 ′ is then sensed by a detector  18 . The detector  18  is fitted with an optical device  19  that can reproduce the fluorescence signal  15 ′ onto a light sensitive sensor  27 . 
   In lieu of the typical large-area lens element, the collimator in the optical device  19  is an array of optic fibers  20 . The optical fiber array catches the light from the wells  12  reflected by the beam splitter  17  by means of the mutually spaced light input areas  21  and harnessing optic fibers so as to transmit the light through parallel harness ends at  23 . 
   Contrary to a collimator composed of lens elements, the collimator configuration of a bundle of optic fibers  20  offers the feature that the light exiting the bundle optic fibers at  23  is collimated more narrowly. That feature is especially advantageous when, for instance, interference filters offering a spectral transfer function that depends on the light&#39;s angle of incidence are configured subsequently. 
   Next, the fluorescence signal  15 ′ is reproduced through the optic fibers harness  23 , through a lens element  24 , through a long wavelength filter  25 , and a further lens element  26  onto a sensor  27 . The long wave pass filter  25  is required in order to filter any shorter wavelength regions out of the excitation light. 
   The design of the shown embodiment mode of the present invention is comparatively simple and economical. 
   In the event that several fluorescence indicators in the PCR batch must be tested, a further beam splitter may be configured in the region of the optical devices  19 , for instance, after the long wavelength filter  25 , to transmit one of the two fluorescence signals while reflecting the other. If one photo-sensitive sensor is mounted in the corresponding light paths, then two or even more fluorescence signals may be processed with further beam splitters. 
   Where several fluorescence indicators are present, a bandpass filter may be additionally present before the sensor in order to transmit the emission range of the particular indicator and to block that of the other indicators. Conceivably as well, and for the purpose of limiting the number of required sensors, exchangeable bandpass filters may be used. 
   To compensate for thermal effects, the apparatus of the present invention may comprise a reference light path running analogously to the path  15 ,  15 ′ except that the LED associated to the reference light path does not illuminate a PCR batch, but rather a reference surface. The light reflected from the surface is analyzed by the detector and the variations incurred during PCR are used to correct the test values. 
   In the above shown embodiment of the present invention, the light sources are the LEDs  14 . However, other appropriate light source may also be used. 
   In the above discussed embodiment, the specimens to be photometrically tested are configured in the wells  12  of a thermocycler  11 . However, the apparatus of the invention also may be used when testing other specimens, illustratively in the form of micro-array spots on a glass plate. In this case, too, it will be the fluorescence effect, which, as a rule, will be utilized. 
   However, optical effects other than the fluorescence effect also may be photometrically determined in tested specimens, for instance transmission, light scattering, and the like. For that purpose, the light paths may be modestly modified but the basic design of the above shown embodiment mode will be preserved. 
   As shown by the single appended drawing, the light sources in the form of the LEDs  14  are individually connected by leads  30  to a control unit  31 . In the state of the art, the control unit would individually drive the LEDs  14  such that only one of the LEDs  14  would be illuminated at a given time. In this case the sensor  27  only receives the light from one specimen of those configured in the wells  12 . In the conventional case, an analyzer  32  following the sensor  27  and illustratively synchronized through a line  23  may unambiguously associate the received light with one of the wells  12 . 
   However, this known timed control of the LEDs  14  entails the drawback that the wells  12  are irradiated at different times over only very short time intervals. 
   Accordingly, in the present invention, the control unit  31  is designed such that all diodes  14  are driven simultaneously whereby the analyzer  32  constantly receives signals from all wells  12  and is able to monitor the signals over a long time interval or, illustratively, may integrate the test values of the individual specimens into a mean value. 
   In order for the analyzer to discriminate between the individual specimens in the wells  12 , the control unit is designed to modulate the light of all diodes  14  by means of different signals. Modulation is preferably amplitude modulation and the applied modulation signals are preferably square. This feature allows simple digital generation and digital analysis of the signals. Instead of the shown control unit  31 , separate individual control units also may be used, which are individually associated with the LEDs  14 . The LEDs  14  may be driven individually or, in special cases, in groups. 
   In one preferred embodiment, the individual signals differ in their frequencies. Accordingly, each LED  14  is modulated at a different frequency. 
   If the analyzer  32  knows the frequencies being used (for instance being permanently programmed into it), then the analyzer shall be able, by means of known algorithms, to filter the individual signals out of the summed light signal from all wells  12  acquired by the sensor  27 , for instance by use of appropriate frequency filters. 
   However, the frequencies are preferably communicated from the control unit  31  through the cable  33  to the analyzer  32 . Especially in the case of many frequencies close to one another, the best approach is to generate the frequencies in the analyzer  31  from a master clock and a specific algorithm (for instance a frequency division procedure). The timing from the master clock may then be applied through the cable  33  to the analyzer  32 , which is able to generate the frequencies according to the same algorithm. In this manner optimal synchronization may be attained. 
   An appropriate way to filter the signals from the various wells  12  out of the summed signal of all specimens picked up by the sensor  27  consists in subjecting the summed signal in the analyzer  32  by means of appropriate circuitry to a Fourier analysis and then to retrieve the amplitudes corresponding to the signal of the particular wells  12  at the various frequencies of the individual signals. 
   When all LEDs  14  are driven simultaneously, a signal may be modulated on all LEDs that will differ for each LED from the signal of the other LEDs by the code sequence. The most appropriate square signals for this purpose for instance may be 1-1-0-1-1-0-1 . . . or 1-0-1-1-0-0-0-1,,, Advantageously, especially with respect to a very large number of specimens or wells  12 , the signals shall be mutually orthogonal. The signals may be repeated at different code sequences in a fixed cycle. The signals also are being applied by the control unit  31  through the line  33  to the analyzer  32 , which multiplies the summed signal of the sensor  27  from the different codes by the individual codes and, in this manner, is able to determine the amplitudes from the particular wells  12 .