HK1097042B - Method and system for distinguishing between materials having overlapping spectra - Google Patents

Description

Method and system for discriminating between materials having overlapping spectra

Technical Field

The present invention relates generally to methods and systems for discriminating between materials having overlapping spectra. Certain embodiments relate to a computer-implemented method that includes determining which material is associated with a ratio between output signals generated by detecting spectra of a single event in two or more detection windows.

Background

Spectroscopic techniques are widely used in the analysis of chemical and biological systems. Most of these techniques involve the absorption and emission of electromagnetic radiation by the material of interest. In most cases, the entire relevant portion of the spectrum under investigation is scanned at a slow rate to provide the most accurate measurement of absorption and emission. However, in other systems, only a particular portion of the spectrum need be examined in order to qualify or quantify the parameter under consideration. Such inspection may be used, for example, if the number of samples is relatively large or the samples must be inspected relatively quickly. In this case, the use of a small "snapshot" of the spectrum can increase the throughput of the sample by reducing the amount of raw data that is processed and analyzed.

One such application is in the field of microarrays (microarrays), a technique used by a large number of disciplines, including the combined chemical and biological assay industry. One company, Luminex ltd, austin, texas, has developed a system for performing bioassays on the surface of various colored fluorescent microspheres. An example of such a system is shown in U.S. patent No. 5,981,180 to Chandler et al. The references are included as if fully set forth herein. The microspheres are interrogated in a fluid flow device by laser excitation and fluorescence detection of each individual microsphere as they pass through a detection zone at a relatively high velocity. With each microsphere emitting several distinct, detectable signals, such a system is capable of analyzing thousands of microspheres in a second. It is clear that processing the entire spectrum and interpreting and decoding the signals from each of the thousands or millions of microspheres will produce an unmanageable amount of data. However, the system described by Chandler et al achieves management of the data only by detecting fluorescence in a specific "window" which is a relatively short (e.g., about 20nm to 40nm), continuous portion of the entire spectrum emitted from the microspheres. Thus, rather than generating the entire fluorescence spectrum for each microsphere, the system generates only a single value for each window (which correlates to the intensity of the signal). These values can be easily exported to a database for further analysis.

In the above systems, the fluorescent dye is absorbed into the microsphere and/or bound to the surface of the microsphere. The dye is selected based on its ability to emit light at the wavelength of the selected window. In addition, the multiple windows are spaced apart and the dye is designed to minimize and preferably eliminate overlap of the fluorescent signals in adjacent windows. By using two windows and two dyes, each at 10 different concentrations, there will thus be 100 fluorescently distinguishable microsphere sets.

Another example of an assay is shown in U.S. patent No. 4,717,655 to Fulwyler, which is incorporated by reference as if fully set forth herein. In particular, Fulwyler describes a method of discriminating between multiple subpopulations of cells (cells) containing labeled particles using two or more labeling reagents. The particles are labeled with a plurality of different preselected ratios of reagents each ranging between zero percent and one hundred percent. Each such reagent has a distinctive, quantifiable signature. In other words, each fluorescent dye has a distinct emission and/or excitation spectrum in a specifically designed color band. The differently labeled particles are mixed with cells suspected of having a specific receptor for the differently labeled particles. Each cell is analyzed to determine the ratio of any two identifiable labeling features associated with each cell such that if its labeling feature ratio correlates with the ratio of preselected labeling agents then it is classified in a subpopulation category. Thus, the method uses ratios to distinguish differently labeled particles by detecting signals from each of the two dyes.

In any of the above systems or methods, there are several ways in which the number of distinguishable groups can be expanded. Using different sized microspheres that can be distinguished based on light scattering will effectively double the number of sets. Another approach is to increase the amount of intensity that each dye can discern. For example, if 15 dye intensities were possible instead of 10 in the example, 225 sets would be obtained. A third method would increase the third window, and then the third dye, or even more, which would exponentially increase the number of sets. Each of these methods has been successfully tested and used to varying degrees. However, each adds a layer of complexity to the system, which can greatly increase the cost or difficulty of creating the platform.

Disclosure of Invention

The present invention relates generally to methods of discriminating between two or more unique but similar spectra. Some embodiments include detecting signals in two or more different detection windows common to two or all spectra. The methods described herein can be used to discriminate between populations of particles exhibiting characteristics of these different spectra and will find utility in many fields, including clinical bioassays.

One embodiment relates to a computer-implemented method that includes determining a ratio between output signals generated by detecting spectra for a single event in two or more detection windows. As used herein, the term "event" is defined as a sample or portion of a sample that is measured to produce an output signal containing meaningful information. In the context of clinical bioassays, an event may be a microsphere, a particle, or a cell as it flows through the measurement window of a fluid flow optical device (e.g., a flow cytometer type instrument). Clearly, there are many other samples or sample portions that can be described by the term "event," and the term "event" as used herein is intended to encompass all possible alternatives.

The term "detection window" as used herein generally relates to a range of wavelengths or 1 wavelength at which an output signal can be generated. The wavelength or range of wavelengths may be determined by the wavelength of the illumination source used to illuminate the material and/or the wavelength of light that can be detected by a detector configured to detect light emitted, scattered, and transmitted by the material. More generally, however, the wavelength of the detection windows will vary depending on the materials being discriminated between them and their respective spectra. For example, the spectra are characteristic of different materials. Furthermore, at least a portion of the spectrum preferably overlaps at least one of the two or more detection windows. The method also includes determining which of the different materials is associated with the ratio. In some embodiments, the method may include determining a concentration of a different material associated with the ratio.

In one embodiment, the two or more detection windows span different continuous portions of the entire spectrum of different materials. Furthermore, each of the output signals may have a single value corresponding to the intensity of the spectrum detected in the respective detection window.

In one embodiment, the two or more detection windows comprise detection windows of different detectors. In an alternative embodiment, the two or more detection windows comprise different detection windows of one detector. In some embodiments, the spectra have peaks at about the same wavelength. Alternatively, the spectra have peaks at different wavelengths. In either embodiment, one of the two or more detection windows may be located entirely within another of the two or more detection windows.

Spectra may be generated as a result of light emitted, absorbed, or transmitted by different materials. In some embodiments, the spectra are generated as a result of fluorescence emitted by different materials. In another embodiment, the different material comprises a material associated with a microsphere. In such an embodiment, the spectra may include different fluorescence emission spectra of the material. In yet another embodiment, the different materials may include multiple materials in solution. In such an embodiment, the spectrum may include different absorption, transmission, and emission of the material. In yet another embodiment, the spectra may include a combination of spectra of two or more materials in solution. In this embodiment, the method may include determining the individual concentrations or ratios of two or more materials in the solution. In one embodiment, the output signal may be generated by a fluid flow device (e.g., a flow cytometer type instrument). In other embodiments, the output signal may be generated by spectroscopic techniques. Each of the embodiments of the method described above may include any other steps described herein.

An additional embodiment relates to another computer-implemented method. The method includes determining a ratio between output signals generated by detecting spectra of a single event in two or more detection windows. This spectrum is characteristic of different materials. At least a portion of the spectrum overlaps in at least one of the two or more detection windows. The method also includes determining the concentration of the one or more different materials by comparing the ratio to a known ratio of a substantially pure sample of individual ones of the different materials. In one embodiment, the different materials are mixed. In another embodiment, the spectra are detected substantially simultaneously.

In one embodiment, the two or more detection windows comprise detection windows of different detectors. In various embodiments, the two or more detection windows comprise different detection windows of one detector. The two or more detection windows span different continuous portions of the entire spectrum of different materials. In some embodiments, the spectra have peaks at approximately the same wavelength. In other embodiments, the spectrum has peaks at different wavelengths. In an additional embodiment, one of the two or more detection windows may be located entirely within another of the two or more detection windows.

In one embodiment, the spectra are generated as a result of fluorescence emitted by different materials. Alternatively, the spectra may be generated as a result of emission, absorption or transmission of different materials. In one embodiment, the different materials comprise materials associated with microspheres, and the spectra comprise different fluorescence emission spectra of the materials. In another embodiment, the different materials may include materials in solution, and the spectra may include different absorption, transmission, or emission spectra of the materials.

In some embodiments, each of the output signals has a single value corresponding to the intensity of the spectrum detected in the respective detection window. In one embodiment, the output signal is generated by a fluid flow optical device. In various embodiments, the output signal is generated by a spectroscopic technique. Each embodiment of the method described above may include any other step described herein.

Another embodiment relates to a different computer-implemented method that includes determining a ratio between output signals generated by detecting spectra for a single event in two or more detection windows. At least a portion of the spectrum overlaps in at least one of the two or more detection windows. The method also includes discriminating between spectra according to the ratio. The method may also include any other steps described herein.

The method described herein advantageously provides a means for two or more distinct but similar spectra to be resolved from each other. Thus, the method can also distinguish between different materials having similar spectra as described above. As such, the method increases the number of dye materials that can be used in the measurement method since dye materials with similar spectra can be distinguished from each other using the methods described herein. Additional advantages of the methods and systems described herein will be apparent from a reading of the detailed description provided below.

An additional embodiment relates to a system that includes one or more detectors and a processor. The one or more detectors are configured to detect spectra of a single event in two or more detection windows. This spectrum is characteristic of different materials. At least a portion of the spectrum overlaps in at least one of the two or more detection windows. The one or more detectors are also configured to generate an output signal in response to the detected spectrum. The processor is configured to determine a ratio between the output signals. The processor is also configured to determine which of the different materials is associated with the ratio. In one embodiment, the processor is further configured to determine a concentration of a different material associated with the ratio.

The two or more detection windows span different continuous portions of the entire spectrum of different materials. In one embodiment, one of the two or more detection windows may be located entirely within another of the two or more detection windows. Furthermore, each of the output signals may have a single value corresponding to the intensity of the spectrum detected in the respective detection window.

In one embodiment, the two or more detection windows may comprise detection windows of different detectors. In another embodiment, the two or more detection windows comprise different detection windows of one detector. In some embodiments, the spectra have peaks at about the same wavelength. In other embodiments, the spectrum has peaks at different wavelengths.

Spectra may be generated as a result of light emitted, absorbed, or transmitted by different materials. In one embodiment, the spectra are generated as a result of fluorescence emitted by different materials. In another embodiment, the different material comprises a material associated with a microsphere. In one such embodiment, the spectra include different fluorescence emission spectra of the material. In one embodiment, the system is configured as a fluid flow device. In another embodiment, the system is configured to perform a spectroscopic technique. The system may be further configured as described herein.

Drawings

Other objects and advantages of the present invention will become more apparent upon reading the following detailed description and upon reference to the accompanying drawings in which:

FIG. 1 is an example of a system for performing the methods described herein.

While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.

Detailed Description

Note that the following description generally describes a technique for interpreting spectra. The following description will generally use fluorescent and fluorescent microspheres in fluid flow optics as examples of applications of the principles. However, the examples provided herein are not intended to limit the use of this technology. For example, it will be apparent to those skilled in the art that this is a technique that is not limited to fluorescent, particle, or fluid flow devices. Suitable microspheres, beads and particles are described in U.S. patent No. 5,736,330 to Fulton, U.S. patent No. 5,981,180 to Chandler et al, U.S. patent No. 6,057,107 to Fulton, U.S. patent No. 6,268,222B1 to Chandler et al, U.S. patent No. 6,449,562B1 to Chandler et al, U.S. patent No. 6,514,295B1 to Chandler et al, U.S. patent No. 6,524,793B1 to Chandler et al, U.S. patent No. 6,528,165B2 to Chandler et al, the disclosures of which are incorporated herein by reference as if fully set forth. The methods described herein may be used with any of the microspheres, pellets, and particles described in these patents. In addition, microspheres used in flow cytometers are available from manufacturers such as Luminex Inc. of Austin, Tex.

Other spectroscopic techniques in which the measured parameter shows a reproducible distribution within a range may also be used such as infrared, ultraviolet/visible (UV/Vis), Raman (Raman), Nuclear Magnetic Resonance (NMR), radioactive emissions, etc. Also, the detectable parameter may be an emission coefficient, an absorption coefficient, a transmission coefficient, or the like. Detection of the signal may be accomplished by any suitable means, including, but not limited to, photomultiplier tubes, avalanche photodiodes, charge coupled devices, pin-hole diodes, and the like. Further to particles, the medium may be a solid, liquid, gas, or any other form in which a signal of the type described herein can be observed.

In conventional spectroscopy, such as fluorescence spectroscopy for example, the wavelength of the source or detector is varied over a range of wavelengths to produce a continuous spectrum of the material being examined. Where the properties of the material are known, but it is desired to detect its presence or concentration, an alternative approach is to keep the excitation and detection wavelengths constant and record the resultant signal. This step produces a single value of the signal due to those particular conditions. A disadvantage of this analysis method is that similar materials, even though their full spectra are clearly different, can display a signal in the spectral "window" being monitored.

An example of a signal that may be similar in the spectral window is the fluorescent signal emitted by Rhodamine (Rhodamine) B and Rhodamine 6G. The former has a peak emission at 543nm, while the latter has a peak emission at 524 nm. If the detection window is set to monitor the spectral region from 520nm to 550nm, both dyes will show a signal of particular significance when excited by the appropriate wavelength. If it is known which dye is being observed, it is possible to determine the concentration of the dye in solution based on the observed signal. However, this signal will not provide a way to distinguish between them if it is not known which dye is in the system. More spectral information is needed to make this distinction.

Using an additional detection window will make it possible to distinguish between two similar spectra. As described above, the detection window spans different continuous portions of the overall spectrum of different materials. However, unlike the previous methods and systems, in the methods and systems described herein, the spectra of multiple materials overlap in at least one of two or more detection windows. For example, returning to the previous example citation, the addition of a detection window configured to detect a spectrum from about 550nm to about 560nm would provide sufficient information to distinguish between dyes and thereby determine concentration. Since each dye emits a broad signal, each dye will have a portion of the signal in this new window. Calculating the ratio R of the second window (550nm to 560nm) signal to the first window (520nm to 550nm) signal will show that R for rhodamine B is greater than R for rhodamine 6G. Importantly, it was observed that the ratio of specific dyes will be somewhat constant over a wide range of dye concentrations. The effectiveness of this technique requires that the spectra of the dyes be sufficiently different that the observed ratios can be consistently distinguished over the working range of concentrations.

Thus, in general, at least a portion of the spectra, which are characteristic of different materials, preferably overlap in at least one of the two or more detection windows. The output signals of the detection windows may have a single value corresponding to the intensity of the spectrum detected in the respective detection window. In this manner, the ratio between the output signals generated by detecting spectra of a single event in two or more detection windows can be used to determine which of the different materials is associated with that ratio. The output signal may be generated by a fluid flow optical device such as the system described herein. Alternatively, the output signal may be generated by a spectroscopic technique including any such technique known in the art.

Furthermore, it is important to note that the system is not limited to discriminating between only two dyes (or other absorbing and reflecting materials). The number of dyes that can be discerned by a set of two windows can be extended by optimizing the position and width of the windows, along with judicious choice of dyes. The ability to produce a unique ratio between the observed signals provides discrimination between different spectral emissions. Also, the signal from the window may be zero for one dye as long as the other dye has a greater signal than zero in this window, since each dye will produce a unique ratio. Similarly, the methods described herein are not limited to only two signal detection windows. Although additional windows will increase the amount of data processed, improved spectral discrimination can be achieved by using additional windows. Furthermore, the size of the spectrum of the detection window may differ from those given as examples. For example, the size of the detection window is limited only by the efficiency of the detection system to define a range and reproducibly measure the signal in that range.

It is also important to note that two or more spectra may or may not deviate with respect to their peaks (e.g., peak intensities). For example, if the emission spectra of two fluorescent dyes show peaks at about the same wavelength, but one peak is wider than the other, then these differences in the spectra can result in a unique intensity ratio between the signals in the two detection windows. In these or other embodiments, one of the two or more detection windows may or may not be located entirely within another of the two or more detection windows.

Further examples illustrate the application of the methods described herein. In the case of the aforementioned fluorescently dyed microspheres, 100 spectrally distinct sets of microspheres can be produced by dyeing a population of microspheres with two different fluorescent dyes (designated herein as a and B) having minimal spectral overlap. One such example is shown in U.S. patent No. 6,514,295 to Chandler et al, the reference of which is incorporated herein as if fully set forth. If two solutions are prepared with 10 different concentrations of each of the two dyes, then 100 different staining solutions and 100 populations of fluorescently different microspheres can be produced. These unique microspheres can be spectrally distinguished using a flow cytometer or other device capable of spectrally interrogating individual microspheres or groups of microspheres.

Microspheres can be spectrally distinguished by detecting the emission signal in a narrow wavelength band window (e.g., in two windows, designated herein as window 1 and window 2). In addition, window 1 is selected to effectively detect the signal from dye a, and window B is selected to correspond to the emission from dye B. If a third window (i.e., window 3) is added that is spectrally close to window 2, then a third dye (dye B') that also emits a signal detectable by window 2 and window 3 can be selected and result in a unique ratio between window 2 and window 3. Then, by establishing 100 new dye solutions using 10 different concentrations of each of dyes a and B', 100 new populations of fluorescent microspheres can be established. It can be readily seen that the addition of another dye (dye B ") that produces a unique ratio between the colors from window 2 and window 3, the use of dyes a and B" will constitute an additional 100 unique clusters of beads. Additional clusters can be generated in a similar manner, as long as it can be found that a unique ratio of dyes will be generated. Furthermore, if another window (window 4) is added, spectrally close to window 1, a similar series can be constructed using new dyes (dyes a ', a ", etc.) in combination with dyes B, B', B", etc., to greatly expand the potential number of unique sets of fluorescent microspheres.

In another embodiment, the method can be used to despin (deconvoltate) overlapping spectra that are determined substantially simultaneously, such as may be seen in a mixed presentation of the complex. For example, the materials attached to the microspheres can be identified as described above. In one such embodiment, the method may include determining the concentration of the one or more different materials by comparing the ratio to a known ratio for a substantially pure sample of the individual materials suspected of comprising the different materials. However, the concentration may be determined from the ratio using any other method known in the art. In this manner, the method may be used to identify microspheres in a multi-information (multi-reporter) assay using two or more dyes on the surface of the sphere, where the determined dye concentration is used to determine the identity of the microspheres. Such materials may also include, for example, fluorescent agents attached in some manner to nucleic acids, enzymes, antigens, and the like. Another example is a solution of two or more dyes that is desired to detect or determine the concentration or concentration ratio of each dye. In particular, the spectra may include spectra of different materials in solution. In such an embodiment, the spectra may include different absorption, transmission, and emission spectra of the material. Further, the spectra may include a combination of spectra of two or more materials in solution. The method may also include determining separate concentrations or ratios for two or more materials in the solution.

In such an example as described above, using a fluorescent dye as an example, if dye a by itself produces a ratio of 1 between the two detection windows and dye B by itself produces a ratio of 100, then the mixing of the two dyes will produce a ratio of between 1 and 100. Furthermore, if the mixture consists mainly of dye a, the observed ratio will be closer to 1; and if there is more dye B, the ratio will be closer to 100. Thus, the ratio will be related to the components of the dye mixture, so that for a particular ratio, it will be possible to calculate the proportion of the observed signal due to each dye composition. Thus where the portion of the signal attributable to each dye is known, and the correlation of the signal to the concentration of the dye is known, then the concentrations of both dyes can be determined simultaneously. New problems may arise from interactions between dyes such as quenching and other energy transfer phenomena, but these problems can be solved by constructing a standard curve using a combination of known dye concentrations. The complexity of analyzing spectra of three or more overlapping dye mixtures may increase significantly, potentially leading to several potential solutions. However, it is possible to remove the incorrect solution by other means.

FIG. 1 shows one example of a measurement system that may be used to perform the methods described herein. Note that figure 1 is not drawn to scale. In particular, the scale of some of the elements of the figures has been greatly exaggerated to emphasize characteristics of the elements.

In fig. 1, the measurement system is shown along a plane of a cross-section of cuvette 12 through which microspheres 10 flow. In one embodiment, the cuvette may be a standard quartz cuvette such as those used in standard flow cytometers. However, any other suitable type of viewing or transport chamber may be used to transport the sample for analysis. The measurement system includes a light source 14. Light source 14 may include any suitable light source known in the art, such as a laser. The light source 14 may be configured to emit light having one or more wavelengths, such as blue or green light. Light source 14 may be configured to illuminate the microspheres as they flow through the cuvette. The illumination may cause the microspheres to emit fluorescent light having one or more wavelengths or wavelength bands. In some embodiments, the system may include one or more lenses (not shown) configured to focus light from the light source onto the microspheres or flow path. The system may also include more than one light source. In one embodiment, the light source may be configured to illuminate the microspheres with light having different wavelengths (e.g., blue and green light). In some embodiments, the light source may be configured to illuminate the microspheres in different directions.

Light scattered forward from the microspheres may be directed to a detection system 16 by a folding mirror or another light directing component. Alternatively, detection system 16 may be placed directly in the forward scatter path. As such, a folding mirror or other light directing component may not be included in the system. In one embodiment, as shown in FIG. 1, the forward scattered light may be light scattered by the microspheres at an angle of about 180 degrees from the direction of illumination by the light source 14. The angle of the forward scattered light may not be exactly 180 degrees from the direction of illumination by the light source 14 so that the incident light from the light source is not incident on the photosensitive surface of the detection system. For example, the forward scattered light may be light scattered by the microspheres at an angle less than or greater than 180 degrees from the direction of illumination by the light source 14 (e.g., light scattered at an angle of about 170 degrees, about 175 degrees, about 185 degrees, or about 190 degrees).

Light scattered and/or emitted by the microspheres at an angle of about 90 degrees from the direction of illumination by the light source 14 may also be collected. In one embodiment, this scattered light may be split into more than one beam by one or more beam splitters or dichroic mirrors. For example, light scattered at an angle of about 90 degrees from the direction of illumination from the light source 14 may be split into two different beams by the beam splitter 20. The two different beams are again split by beamsplitters 22 and 24 to produce four different beams. Each beam may be directed to a different detection system, which may include one or more detectors. For example, one of the four beams may be directed to detection system 26. Detection system 26 may be configured to detect light scattered by the microspheres.

The other three beams of light may be directed to detection systems 28, 30, and 32. Detection systems 28, 30, and 32 may be configured to detect fluorescent light emitted by the microspheres. Each detection system may be configured to detect fluorescence at a different wavelength or different wavelength range. For example, a detection system may be configured to detect green fluorescence. Another detection system is configured to detect orange fluorescence. A further detection system is configured to detect red fluorescence. In another embodiment, different detectors have different detection windows, at least a portion of the spectra of different materials overlapping in at least one of the detection windows as further described above. In a different embodiment, one of the detectors may have different detection windows in one of which at least a portion of the spectra of different materials overlap. One example of a detector that may have multiple detection windows is a multi-anode photomultiplier tube, where each anode may serve as a different detection window.

In some embodiments, spectral filters 34, 36, and 38 may be coupled to detection systems 28, 30, and 32, respectively. The spectral filter may be configured to block fluorescence other than those wavelengths that the detection system is configured to detect. Further, one or more lenses (not shown) may be optically coupled to each detection system. The lens may be configured to focus the scattered light or the emitted fluorescent light onto a photosensitive surface of the detector.

The output current of the detector is proportional to the fluorescence light incident on it and results in a current pulse. The current pulses may be converted to voltage pulses, low pass filtered, and then digitized by an a/D converter (not shown). A processor 40, such as a DSP, integrates the area under the pulse to provide a value representative of the fluorescence amplitude. Further, the processor may perform other functions described herein (e.g., determining a ratio between the output signals and determining which of the different materials is associated with the ratio). As shown in fig. 1, processor 40 may be coupled to detector 26 via a transmission medium 42. Processor 40 may also be indirectly coupled to detector 26 via a transmission medium 42 and one or more other components (not shown), such as an a/D converter. The processor may be coupled to the other components of the system in a similar manner.

In some embodiments, the output signals generated from the fluorescence emitted by the microspheres may be processed to determine the identity of the microspheres and information about the reactions occurring at the surface of the microspheres. For example, output signals from two or more detectors may be used to determine the identity of the microsphere, and other output signals may be used to determine the reaction occurring on the surface of the microsphere. The identity of the microsphere may be determined based on a ratio of the output signals produced in two or more different windows. For example, if detection systems 30 and 32 have different detection windows, the identity of the microsphere may be determined based on the ratio of the output signal generated by detection system 30 to the output signal generated by detection system 32, along with the intensity of each signal. Thus, the choice of detector and spectral filter will vary depending on the type of dye included in or bound to the microsphere and/or the reaction being measured (i.e., the dye included in or bound to the reactant involved in the reaction).

In a particular embodiment, the choice of detector and/or spectral filter may depend on the peak value of the dye included in or bound to the microsphere. For example, as described above, rhodamine B has a peak emission at 543nm and rhodamine 6G has a peak emission at 524 nm. If the microspheres are dyed with one or both dyes at different concentrations, detection system 30 may be configured to detect light in a wavelength range from about 520nm to about 550 nm. Further, the detection system 32 may be configured to detect light in a wavelength range from about 550nm to about 560 nm.

In another embodiment, as described above, the emission spectra of the two fluorescent dyes may not deviate, but may show peak emissions at about the same wavelength. In such an example, the characteristics of the emission spectrum on either or both sides of the peak emission may be different. Likewise, the emission spectrum has a unique intensity ratio between the signals in the two detection windows. Thus, in one embodiment, although the detection systems 30 and 32 have different detection windows, the detection window of one of the detection systems may be located wholly or partially within the detection window of the other detection system. For example, if the peak emissions of the two dyes are at about 540nm, the detection window of detection system 30 may have a wavelength range from about 530nm to about 550nm, and the detection window of detection system 32 may have a wavelength range from about 510nm to about 570 nm. Note that the above wavelength ranges are only examples and differ according to the dye, e.g. of the microsphere.

Although the system of fig. 1 is shown to include a detection system having two different detection windows for discriminating between microspheres having different dye characteristics, it should be understood that the system may include more than two such detection windows (i.e., 3 detection windows, 4 detection windows, etc.). In such embodiments, the system may include additional beam splitters and additional detection systems with other detection windows. The detection windows for more than two detection systems may be determined as described above. Furthermore, a spectral filter or lens may be coupled to each additional detection system.

In another embodiment, the system may include two or more detection systems configured to discriminate between different materials reacted on the surface of the microsphere. The different reactant materials may have different dye characteristics than the microspheres. However, the reactant materials may have dye characteristics such that they have similar emission spectra. For example, the emission spectra of the reactant materials may overlap. In one embodiment, the emission spectrum may have off-peak emission, but may also exhibit strong signals at one or more of the same wavelengths. In a different embodiment, the emission spectra are on either or both sides of the peak emission, and the emission spectra may have substantially the same peak emission but different characteristics. Thus, the emission spectrum and the reactant material may be distinguished as described above.

Additional examples of measurement systems that can be used to perform the methods described herein are shown in U.S. Pat. No. 5,981,180 to Chandler et al, U.S. Pat. No. 6,046,867 to Chandler, U.S. Pat. No. 6,139,800 to Chandler, U.S. Pat. No. 6,366,354 to Chandler, U.S. Pat. No. 6,411,904 to Chandler, U.S. Pat. No. 6,449,562 to Chandler et al, and U.S. Pat. No. 6,524,793 to Chandler et al, the references of which are incorporated herein as if fully set forth. The systems described in these patents may be configured with a system as described above. The measurement system described herein may also be further configured as described in these patents.

Program instructions implementing methods such as those described herein may be transmitted over or stored on a carrier medium. The carrier medium may be a transmission medium such as a wire, cable, or wireless transmission line, or a signal propagating along such a wire, cable, or line. The carrier medium may also be a storage medium such as read-only memory, random access memory, magnetic or optical disk, or magnetic tape.

In one embodiment, a processor, such as processor 40 of FIG. 1, may be configured to execute program instructions to perform a computer-implemented method according to the above embodiments. The processor may take various forms, including a DSP, a personal computer system, a mainframe computer system, a workstation, a network appliance, an internet appliance, a personal digital assistant ("PDA"), a television system, or other device. In general, the term "computer system" may be broadly defined to include any device having one or more processors capable of executing instructions from a storage medium.

The program instructions may be implemented in any of various ways, including step-based techniques, component-based techniques, and/or object-oriented techniques. For example, the program instructions may be executed as desired using ActiveX controls, C + + objects, JavaBeans, Microsoft Foundation classes ("MFCs"), or other techniques or methods.

It will be appreciated by those skilled in the art having the benefit of this disclosure that the present invention is directed to methods for discriminating between similar absorption, transmission and emission spectra. Modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention herein shown and described are to be taken as the presently preferred embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be altered, certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Claims (46)

1. A computer-implemented method for discriminating between materials having overlapping spectra, comprising:

determining a ratio between output signals generated by detecting spectra of a single event in two or more detection windows, wherein the spectra are characteristic of different materials, and wherein at least a portion of the spectra overlap in at least one of the two or more detection windows; and

determining which of the different materials is associated with the ratio.

2. The method of claim 1, wherein the two or more detection windows comprise detection windows of different detectors.

3. The method of claim 1, wherein the two or more detection windows comprise different detection windows of one detector.

4. The method of claim 1, wherein the spectra have peaks at about the same wavelength.

5. The method of claim 1, wherein the spectra have peaks at different wavelengths.

6. The method of claim 1, wherein the spectra are generated as a result of fluorescence emitted by different materials.

7. The method of claim 1, wherein the two or more detection windows span different contiguous portions of the entire spectrum of different materials.

8. The method of claim 1, wherein each of the output signals has a single value corresponding to the intensity of the spectrum detected in the respective detection window.

9. The method of claim 1, wherein one of the two or more detection windows is entirely located within another of the two or more detection windows.

10. The method of claim 1, wherein the different materials comprise materials associated with microspheres, and wherein the spectra comprise different fluorescence emission spectra of the materials.

11. The method of claim 1, wherein the different materials comprise materials in solution, and wherein the spectra comprise different absorption, transmission, or emission spectra of the materials.

12. The method of claim 1, wherein the spectra comprise a combination of spectra of two or more materials in a solution, the method further comprising determining respective concentrations or ratios of the two or more materials in the solution.

13. The method of claim 1, wherein the output signal is generated by a fluid flow optical device.

14. The method of claim 1, wherein the output signal is generated by a spectroscopic technique.

15. The method of claim 1, wherein the spectra are generated as a result of light emitted, absorbed, or transmitted by different materials.

16. The method of claim 1, further comprising determining a concentration of a different material associated with the ratio.

17. A computer-implemented method for discriminating between materials having overlapping spectra, comprising:

determining a ratio between output signals generated by detecting spectra of a single event in two or more detection windows, wherein the spectra are characteristic of different materials, and wherein at least a portion of the spectra overlap in at least one of the two or more detection windows; and

the concentration of one or more of the different materials is determined by comparing the ratio to a known ratio for a substantially pure sample of each of the different materials.

18. The method of claim 17, wherein the different materials are mixed.

19. The method of claim 17, wherein the spectra are detected simultaneously.

20. The method of claim 17, wherein the two or more detection windows comprise detection windows of different detectors.

21. The method of claim 17, wherein the two or more detection windows comprise different detection windows of one detector.

22. The method of claim 17, wherein the spectra have peaks at about the same wavelength.

23. The method of claim 17, wherein the spectra have peaks at different wavelengths.

24. The method of claim 17, wherein the spectra are generated as a result of fluorescence emitted by different materials.

25. The method of claim 17, wherein the two or more detection windows span different contiguous portions of the entire spectrum of different materials.

26. The method of claim 17, wherein each of the output signals has a single value corresponding to the intensity of the spectrum detected in the respective detection window.

27. The method of claim 17, wherein one of the two or more detection windows is entirely located within another of the two or more detection windows.

28. The method of claim 17, wherein the different materials comprise materials associated with microspheres, and wherein the spectra comprise different fluorescence emission spectra of the materials.

29. The method of claim 17, wherein the different materials comprise materials in solution, and wherein the spectra comprise different absorption, transmission, or emission spectra of the materials.

30. The method of claim 17, wherein the output signal is generated by a fluid flow optical device.

31. The method of claim 17, wherein the output signal is generated by a spectroscopic technique.

32. The method of claim 17, wherein the spectra are generated as a result of light emitted, absorbed, or transmitted by different materials.

33. A system for discriminating between materials having overlapping spectra, comprising:

one or more detectors configured to detect spectra of a single event in two or more detection windows, wherein the spectra are characteristic of different materials, wherein at least a portion of the spectra overlap in at least one of the two or more detection windows, and wherein the one or more detectors are further configured to generate an output signal responsive to the detected spectra; and

a processor configured to determine a ratio between the output signals and to determine which of the different materials is associated with the ratio.

34. The system of claim 34, wherein the two or more detection windows comprise detection windows of different detectors.

35. The system of claim 34, wherein the two or more detection windows comprise different detection windows of one detector.

36. The system of claim 34, wherein the spectra have peaks at about the same wavelength.

37. The system of claim 34, wherein the spectra have peaks at different wavelengths.

38. The system of claim 34, wherein the spectra are generated as a result of fluorescence emitted by different materials.

39. The system of claim 34, wherein the two or more detection windows span different contiguous portions of the entire spectrum of different materials.

40. The system of claim 34, wherein each of the output signals has a single value corresponding to the intensity of the spectrum detected in the respective detection window.

41. The system of claim 34, wherein one of the two or more detection windows is entirely located within another of the two or more detection windows.

42. The system of claim 34, wherein the different materials comprise materials associated with microspheres, and wherein the spectra comprise different fluorescence emission spectra of the materials.

43. The system of claim 34, wherein the system is configured to be produced by a fluid flow optical device.

44. The system of claim 34, wherein the system is configured to perform a spectroscopic technique.

45. The system of claim 34, wherein the spectra are generated as a result of light emitted, absorbed, or transmitted by different materials.

46. The system of claim 34, wherein the processor is further configured to determine a concentration of the different material associated with the ratio.