CN1823269A - Methods and systems for discriminating between materials with overlapping spectra - Google Patents

Abstract

Various computer-implemented methods and systems are provided. A computer-implemented 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 which of the different materials is associated with the ratio. One embodiment of the system includes one or more detectors configured to detect spectra of a single event in two or more detection windows. The spectrum comprises a spectrum as described above. The one or more detectors are also configured to generate an output signal responsive to the detected spectrum. The system also includes a processor configured to determine a ratio between the output signals and determine which of the different materials is associated with the ratio.

Description

Methods and systems for discriminating between materials with overlapping spectra

technical field

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

Background technique

Spectroscopy 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 study is scanned at low speed to provide the most accurate measurements of absorption and emission. In other systems, however, only certain parts of the spectrum need to 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 small spectral "snapshots" can increase sample throughput by reducing the amount of raw data that is processed and analyzed.

One such application is in the field of microarrays, a technology used by a wide variety of disciplines including the combined chemical and biological assay industries. Luminex, Inc., a company in Austin, Texas, has developed a system for performing bioassays on the surface of variously colored fluorescent microspheres. An example of such a system is shown in US Patent No. 5,981,180 to Chandler et al. References thereto are included as if given in full. Microspheres are interrogated in the fluid flow device by laser excitation and fluorescence detection of each individual microsphere as they pass through a detection region at relatively high speed. With each microsphere emitting several clear, detectable signals, such a system can analyze thousands of microspheres a second. Clearly, processing the entire spectrum and interpreting and decoding the signal from each of the thousands or millions of microspheres would generate an unmanageable amount of data. However, the system described by Chandler et al. achieves data management only by detecting fluorescence in a specific "window", which is a relatively short (e.g., about 20 nm to 40 nm) part of the entire spectrum emitted from the microsphere. continuous part. Thus, instead of generating the entire fluorescence spectrum for each microsphere, the system generates only a single value (which correlates to the intensity of the signal) for each window. These values can be easily exported to a database for further analysis.

In the systems described above, fluorescent dyes are absorbed into the microspheres and/or bound to the surface of the microspheres. Dyes are selected based on their ability to emit light at a wavelength of a selected window. In addition, multiple windows are spaced apart, and dyes are designed to minimize and preferably eliminate overlap of fluorescent signals within adjacent multiple windows. By using two windows and two dyes, each at 10 different concentrations, there will be 100 fluorescently discernible sets of microspheres.

Another example of an assay method is shown in US Patent No. 4,717,655 to Fulwyler, the citation of which is incorporated herein as if fully set forth. In particular, Fulwyler describes a method for discriminating subpopulations of cells containing labeled particles using two or more labeling reagents. The particles are labeled with a number of different preselected ratios of reagents ranging from zero percent to one hundred percent for each reagent. Each of these reagents has distinct, quantifiable labeling characteristics. In other words, each fluorescent dye has a distinct emission and/or excitation spectrum in a specifically designed color band. Differently labeled particles are mixed with cells suspected of having specific receptors for the differently labeled particles. Each cell is analyzed to determine the ratio of any two identifiable marker signatures associated with each cell such that it is classified in a subpopulation class if its marker signature ratio correlates with the ratio of preselected marker reagents. Thus, the method utilizes ratios to discriminate differentially labeled particles by detecting signals from each of the two dyes.

In any of the systems or methods described above, there are several ways in which the number of discernable groups can be expanded. Using microspheres of different sizes, which can be discriminated based on light scattering, would effectively double the number of groups. Another approach is to increase the number of discernible intensities of each dye. For example, if 15 dye intensities were possible instead of 10 in the example, then 225 sets would be obtained. A third approach would add a third window, and then a third dye, or even more, which would exponentially increase the number of groups. 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 expense or difficulty of generating the platform.

Contents of the invention

The present invention generally relates to methods of discriminating between two or more unique but similar spectra. Certain 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 characteristic particle populations exhibiting these different profiles and will find utility in many fields including clinical bioassays.

One embodiment relates to a computer-implemented method comprising determining a ratio between output signals produced by detecting a spectrum of 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 that contains meaningful information. In the context of clinical bioassays, an event may be a microsphere, particle, or cell as it flows through the measurement window of a fluid flow optical device (eg, 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 here is intended to encompass all possible alternatives.

The term "detection window" as used herein generally refers to a wavelength or range of wavelengths at which an output signal can be generated. Such a 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 window will vary depending on the materials being discriminated between them and their respective spectra. For example, the spectrum is 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 concentrations of different materials associated with the ratio.

In one embodiment, two or more detection windows span different contiguous portions of the entire spectrum of different materials. Furthermore, each of the output signals may have a single value corresponding to the spectral intensity detected in the corresponding 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 a detector. In some embodiments, the spectra have peaks at about the same wavelength. Alternatively, the spectrum has peaks at different wavelengths. In either embodiment, one of the two or more detection windows may be entirely within the other of the two or more detection windows.

Spectrum can be produced 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 the material associated with the microspheres. In such an embodiment, the spectrum 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 the different absorption, transmission and emission of the material. In yet another embodiment, the spectrum may comprise a combination of the 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 (eg, a flow cytometer type instrument). In other embodiments, the output signal may be generated by spectroscopic techniques. Each embodiment of the methods described above may include any of the other steps described herein.

An additional embodiment relates to another computer-implemented method. The method includes determining a ratio between output signals produced by detecting the 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 ratios to known ratios for substantially pure samples of individual ones of the different materials. In one embodiment, different materials are blended. 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 a detector. Two or more detection windows span different consecutive 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 entirely within the other of the two or more detection windows.

In one embodiment, the spectrum is generated as a result of the fluorescence emitted by the different materials. Alternatively, spectra may be generated as a result of emission, absorption, or transmission by different materials. In one embodiment, the different material includes a material associated with the microspheres, and the spectrum includes a different fluorescence emission spectrum of the material. 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 corresponding detection window. In one embodiment, the output signal is generated by fluid flow optics. In various embodiments, the output signal is generated by spectroscopic techniques. Each embodiment of the methods described above may include any of the other steps described herein.

Another embodiment relates to a different computer-implemented method comprising determining a ratio between output signals produced by detecting a spectrum of 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 based on ratios. The method also includes any other steps described herein.

The method described here advantageously provides a method for two or more distinct but similar spectra to be resolved from each other. Thus, the method can also discriminate between different materials with similar spectra as described above. As such, the method increases the amount of dye material 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 upon reading the detailed description provided below.

An additional embodiment involves a system including one or more detectors and a processor. One or more detectors are configured to detect the spectrum 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 the concentration of the different material associated with the ratio.

Two or more detection windows span different consecutive portions of the entire spectrum of different materials. In one embodiment, one of the two or more detection windows may be entirely within the other of the two or more detection windows. Furthermore, each of the output signals may have a single value corresponding to the spectral intensity detected in the corresponding 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 a detector. In some embodiments, the spectra have peaks at about the same wavelength. In other embodiments, the spectrum has peaks at different wavelengths.

Spectrum can be produced 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 the material associated with the microspheres. In one such embodiment, the spectrum includes distinct 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 spectroscopic techniques. The system may further be configured as described herein.

Description of drawings

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

Figure 1 is a diagram illustrating an example of a system for performing the methods described herein.

While the invention is susceptible to various modifications and alterations, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the drawings and detailed description herein are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all things falling within the spirit and scope of the invention as defined by the appended claims. Modifications, Equivalents and Substitutions.

Detailed ways

Note that the description below roughly describes one technique for interpreting spectra. The following description takes fluorescent and fluorescent microspheres commonly used in fluid flow optics as an example of the application of the principle. However, the examples provided here are not intended to limit the use of this technique. For example, it will be apparent to those skilled in the art that this is a technology that is not limited to fluorescence, particle or fluid flow devices. Suitable microspheres, pellets and particles are described in US Patent No. 5,736,330 to Fulton, US Patent No. 5,981,180 to Chandler et al., US Patent No. 6,057,107 to Fulton, US Patent No. 6,268,222B1 to Chandler et al., 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. here. The methods described herein can use any of the microspheres, pellets and particles described in these patents. Additionally, microspheres for use in flow cytometers are available from manufacturers such as Luminex, Inc. of Austin, Texas.

Other spectroscopic techniques in which the measured parameters show reproducible distributions over a range may also be used such as infrared, ultraviolet/visible (UV/Vis), Raman, nuclear magnetic resonance (NMR), radioactive emission, and the like. Likewise, detectable parameters may be emission coefficients, absorption coefficients, transmission coefficients, and the like. Detection of the signal can be accomplished by any suitable device, including but not limited to, photomultiplier tubes, avalanche photodiodes, charge-coupled devices, pinhole diodes, and the like. Also for particles, the medium may be a solid, liquid, gas, or any other form in which signals of the type described herein can be observed.

In traditional spectroscopy, such as fluorescence spectroscopy, for example, the wavelength of the source or detector is varied over a range of wavelengths to produce a continuum of spectra of the material being examined. When the properties of a material are known, but its presence or concentration is desired to be detected, an alternative is to hold the excitation and detection wavelengths constant and record the resultant signal. This step yields a single value for the signal attributed to those particular conditions. The disadvantage of this method of analysis is that similar materials can show signals in the spectral "window" being monitored even though their full spectra are clearly different.

An example of a signal that may be similar in a spectral window is the fluorescent signal emitted by Rhodamine B and Rhodamine 6G. The former has a peak emission at 543nm, while the latter has a peak emission at 524nm. If the detection window is set to monitor the spectral region from 520nm to 550nm, the two dyes will display a signal of special interest 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, without knowing which dye is in the system, this signal will not provide a way to discriminate between them. More spectral information is needed to make this distinction.

Using an additional detection window will allow the two similar spectra to be distinguished. As mentioned above, the detection windows span different contiguous parts of the full 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 the two or more detection windows. For example, returning to the citation of the previous example, the addition of a detection window configured to detect the spectrum from about 550nm to about 560nm would provide sufficient information to distinguish between dyes, and thus concentration can be determined. Since each dye emits a broad signal, each dye will have a portion of that 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 Rhodamine B has a greater R than Rhodamine 6G. Importantly, it was observed that the ratio of a particular dye 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 distinct that the observed ratios can be consistently discerned over the working range of concentrations.

Therefore, in general, at least part of the spectrum, which is characteristic of different materials, preferably overlaps in at least one of the two or more detection windows. The output signal of a detection window 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 produced by detecting the spectra of a single event in two or more detection windows can be used to determine which of the different materials is associated with the ratio. The output signal may be generated by fluid flow optics such as the systems described herein. Alternatively, the output signal may be generated by spectroscopic techniques 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 discernible by a set of two windows can be expanded by optimizing the position and width of the windows along with judicious choice of dyes. The ability to generate a unique ratio between the observed signals provides for discrimination of different spectral emissions. Also, since each dye will produce a unique ratio, the signal from a window can be zero for one dye as long as the other dye has a signal greater than zero in this window. Similarly, the methods described herein are not limited to only two signal detection windows. Although additional windows will increase the amount of data processed, increased spectral discrimination can be achieved through the use of additional windows. Furthermore, the dimensions 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 the range and reproducibly measure signals in that range.

It is also important to note that two or more spectra may or may not deviate with respect to their peaks (eg, peak intensities). For example, if the emission spectra of two fluorochromes exhibit peaks at about the same wavelength, but one peak is wider than the other, these differences in the spectra can result in unique intensities between the signals in the two detection windows. ratio. In these or other embodiments, one of the two or more detection windows may or may not all be within the other of the two or more detection windows.

A further example shows the application of the methods described here. In the aforementioned case of fluorescently stained microspheres, 100 spectrally distinct groups of microspheres could be generated by staining populations of microspheres with two different fluorescent dyes (designated here as A and B) with minimal spectral overlap. One such example is shown in US Patent No. 6,514,295 to Chandler et al., the citation of which is incorporated herein as if given in its entirety. If two solutions were prepared with 10 different concentrations of each of the two dyes, 100 different staining solutions and 100 populations of microspheres with different fluorescence could be produced. These unique microspheres can be identified spectroscopically using a flow cytometer or other device capable of spectrally interrogating individual microspheres or groups of microspheres.

Microspheres can be spectrally discriminated by detecting emission signals in a narrow wavelength band window (eg, in two windows, designated here as window 1 and window 2). Furthermore, window 1 was selected to efficiently detect the signal from dye A, and window B was selected to correspond to the emission from dye B. If a third window (i.e., window 3) is added, which is spectrally close to window 2, then a third dye (dye B') that also emits a signal detectable by windows 2 and 3 can be selected and results in window Unique ratio between 2 and window 3. Then, by creating 100 new dye solutions using 10 different concentrations of each of dyes A and B', 100 new populations of fluorescent microspheres can be created. It can be easily seen that adding another dye (dye B") that produces a unique ratio between windows 2 and 3, using dyes A and B" will constitute an additional 100 unique populations of globules. Additional populations can be generated in a similar manner as long as dyes can be found that will generate unique ratios. Furthermore, if another window (window 4) is added, which is spectrally close to window 1, a similar series can be formed using new dyes (dye A', A", etc.) in combination with dyes B, B', B", etc. , to greatly expand the potential number of unique groups of fluorescent microspheres.

In another embodiment, the method can be used to deconvolute overlapping spectra that are determined substantially simultaneously, such as can be seen when blending of complexes occurs. For example, materials attached to 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 ratios to known ratios of a sufficiently pure sample of individual materials that are suspected to constitute the different materials. However, the concentration can be determined from a ratio using any other method known in the art. As such, the method can be used to identify microspheres in a multi-reporter assay using two or more dyes on the surface of the sphere when defined dye concentrations are used to determine microsphere identity. 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 where it is desired to detect or determine the concentration or concentration ratio of each dye. In particular, the spectra may comprise spectra of different materials in solution. In such an embodiment, the spectrum may include the different absorption, transmission and emission spectra of the material. Additionally, a spectrum may include a combination of the spectra of two or more materials in solution. The method may also include determining separate concentrations or ratios of two or more materials in solution.

In such an example as described above, using fluorescent dyes as an example, if dye A by itself produces a ratio of 1 between two detection windows, and dye B produces by itself a ratio of 100, then the mixing of the two dyes Will produce a ratio between 1 and 100. Furthermore, the observed ratio will be closer to 1 if the mixture consists mostly of Dye A, and closer to 100 if there is more Dye B. Thus, the ratio will be related to the composition 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 when the portion of the signal attributable to each dye is known, and the dependence of the signal on the dye concentration 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 resolved by constructing a standard curve using known combinations of dye concentrations. The complexity of analyzing the spectra of a mixture of three or more overlapping dyes can increase significantly, potentially yielding several potential solutions. However, it is possible to remove incorrect solutions by other means.

Figure 1 shows an example of a measurement system that can be used to perform the methods described herein. Note that Figure 1 is not drawn to scale. In particular, the scale of some elements of the figures has been greatly exaggerated to emphasize the features of the elements.

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

Light scattered forward from the microspheres can be directed to detection system 16 via a fold mirror or another light directing component. Alternatively, the detection system 16 can be placed directly in the path of the forward scattered light. As such, folding mirrors or other light directing components may not be included in this 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 from the light source 14 . The angle of the forward scattered light may not be exactly 180 degrees from the direction of the light source 14 so that the incident light from the light source does not impinge on the photosensitive surface of the detection system. For example, forward scattered light may be light scattered by microspheres at angles less than or greater than 180 degrees from the direction of illumination from light source 14 (e.g., at about 170 degrees, about 175 degrees, about 185 degrees, or about 190 degrees) light).

Light scattered and/or emitted by the microspheres at an angle of approximately 90 degrees from the direction of illumination from 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 approximately 90 degrees from the direction irradiated by the light source 14 may be split into two different beams by the beam splitter 20 . These two different beams are split again by beam splitters 22 and 24 to generate four different beams. Each beam can be directed to a different detection system, which can include one or more detectors. For example, one of 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 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 can be configured to detect fluorescence at a different wavelength or range of wavelengths. For example, a detection system can be configured to detect green fluorescence. Another detection system is configured to detect orange fluorescence. Yet another detection system is configured to detect red fluorescent light. In another embodiment, the different detectors have different detection windows, at least one of the detection windows as described further above, at least part of the spectrum of the different materials overlaps. In a different embodiment, one of the detectors may have different detection windows, in one of the detection windows at least part of the spectra of different materials overlap. An example of a detector that can have multiple detection windows is a multi-anode photomultiplier tube, where each anode can be used 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 filter may be configured to block fluorescence at wavelengths other than those wavelengths that the detection system is configured to detect. Additionally, one or more lenses (not shown) may be optically coupled to each detection system. The lens may be configured to focus scattered light or emitted fluorescence onto the photosensitive surface of the detector.

The output current of the detector is proportional to the fluorescence incident on it and results in a current pulse. The current pulses can 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 magnitude of the fluorescence. Additionally, the processor can perform other functions described herein (eg, determining a ratio between 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 transmission medium 42 . Processor 40 may also be indirectly coupled to detector 26 via transmission medium 42 and one or more other components (not shown), such as an A/D converter. The processor can be coupled to other components of the system in a similar manner.

In some embodiments, output signals generated from the fluorescence emitted by the microspheres can be processed to determine the identity of the microspheres and information about reactions occurring on the surface of the microspheres. For example, output signals from two or more detectors can be used to determine the identity of the microsphere, and other output signals can be used to determine the reaction occurring on the surface of the microsphere. The identity of the microspheres can be determined based on the ratio of output signals generated in two or more different windows. For example, if detection systems 30 and 32 have different detection windows, the identity of the microspheres can 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 detectors and filters varies depending on the dye included in or bound to the microsphere and/or the type of reaction being measured (i.e., the dye included in or bound to the reactants involved in the reaction). .

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

In another embodiment, as described above, the emission spectra of the two fluorochromes may not diverge, but instead may exhibit peak emissions at approximately the same wavelength. In such an example, the characteristics of the emission spectrum may differ on either or both sides of the peak emission. Likewise, the emission spectrum has a unique intensity ratio between the signals in the two detection windows. Thus, in one embodiment, even though detection systems 30 and 32 have different detection windows, the detection window of one of the detection systems may be wholly or partially within the detection window of the other detection system. For example, if both dyes have peak emissions at about 540 nm, the detection window of detection system 30 may have a wavelength range from about 530 nm to about 550 nm, and the detection window of detection system 32 may have a wavelength range from about 510 nm to about 570 nm. Note that the above wavelength ranges are examples only and vary depending on, for example, the dye of the microspheres.

Although the system of FIG. 1 is shown as including a detection system with two different detection windows for discriminating between microspheres with different dye properties, 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 beamsplitters with other detection windows and additional detection systems. Detection windows for more than two detection systems may be determined as described above. In addition, spectral filters or lenses can 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 reacting on the surface of the microspheres. Different reactant materials may have different dye properties than those of the microspheres. However, the reactant materials may have dye properties 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 emissions, but may also exhibit strong signals at one or more of the same wavelengths. In a different embodiment, the emission spectra being on either or both sides of the peak emission, the emission spectra may have approximately the same peak emission but different characteristics. Thus, emission spectra and reactant materials can be discerned as described above.

Additional examples of measurement systems that may be used to perform the methods described herein are found in Chandler et al., U.S. Patent No. 5,981,180, Chandler U.S. Patent No. 6,046,867, Chandler U.S. Patent No. 6,139,800, Chandler U.S. Patent No. 6,366,354, US Patent No. 6,411,904 to Chandler, US Patent No. 6,449,562 to Chandler et al., US Patent No. 6,524,793 to Chandler et al., which are incorporated herein by reference as if fully given. The systems described in these patents can be configured with systems as described above. The measurement systems described here can also be further configured as described in these patents.

Program instructions such as those described herein implementing methods may be transmitted on 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 a read-only memory, random access memory, magnetic or optical disks, or magnetic tape.

In one embodiment, a processor such as processor 40 of FIG. 1 may be configured to execute program instructions according to the above embodiments to perform the computer-implemented method. The processor may take different forms, including a DSP, personal computer system, mainframe computer system, workstation, network appliance, Internet appliance, personal digital assistant ("PDA"), 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.

Program instructions may be implemented in any number of different ways, including step-based techniques, component-based techniques, and/or object-oriented techniques. For example, program instructions may be executed as desired using ActiveX controls, C++ objects, JavaBeans, Microsoft Foundation Classes ("MFC"), or other technologies or methodologies.

It should be understood by those skilled in the art having the benefit of this disclosure that the present invention is intended to provide methods for discriminating between similar absorption, transmission and emission spectra. Modifications and alternative embodiments of the various aspects of the invention will be apparent to those skilled in the art from this description. Therefore, this specification is to be interpreted as exemplary only and for the purpose of teaching those skilled in the art in an ordinary manner to carry out the present invention. It is to be understood that the form of the invention shown and described herein is considered to be the presently preferred embodiment. Elements and materials shown and described herein may be substituted, parts and procedures may be altered, and certain features of the invention may be utilized in isolation, all to the benefit of one skilled in the art having the benefit of the description of the invention. It is obvious to the skilled person. 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 (47)

1. computer-implemented method comprises:

Definite ratio between output signals that produces by the spectrum of surveying the individual event in two or more detection windows, wherein said spectrum is the feature of different materials, and wherein the described spectrum of at least a portion overlaps at least one of two or more detection windows; And

Determine that any and described ratio in the described different materials is associated.

2. the method for claim 1 is characterized in that, two or more detection windows comprise the detection window of different detectors.

3. the method for claim 1 is characterized in that, two or more detection windows comprise the different detection windows of a detector.

4. the method for claim 1 is characterized in that, described spectrum has peak value at about identical wavelength place.

5. the method for claim 1 is characterized in that, described spectrum has peak value at the different wave length place.

6. the method for claim 1 is characterized in that, produces described spectrum as the result by the different materials emitting fluorescence.

7. the method for claim 1 is characterized in that, the different continuous part of the whole spectrum of described two or more detection windows span different materials.

8. the method for claim 1 is characterized in that, each in the described output signal has the single value corresponding to the spectral intensity that detects in corresponding detection window.

9. the method for claim 1 is characterized in that, in described two or more detection windows one all is arranged in another of described two or more detection windows.

10. the method for claim 1 is characterized in that, described different materials comprises the material that is associated with microsphere, and described spectrum comprises the different fluorescence emission spectrums of described material.

11. the method for claim 1 is characterized in that, described different materials is included in the material in the solution, and described spectrum comprises difference absorption, transmission or the emission spectrum of described material.

12. the method for claim 1 is characterized in that, described spectrum is included in the combination of the spectrum of two or more materials in the solution, and described method also comprises each concentration or the ratio of determining two or more materials in the solution.

13. the method for claim 1 is characterized in that, described output signal is produced by fluid flow optical device.

14. the method for claim 1 is characterized in that output signal is produced by spectral technique.

15. the method for claim 1 is characterized in that, produces described spectrum as the result by different materials emission, absorption or transmitted light.

16. the method for claim 1 is characterized in that, also comprises the concentration of definite different materials that is associated with described ratio.

17. a computer-implemented method comprises:

Definite ratio between output signals that produces by the spectrum of surveying the individual event in two or more detection windows, wherein said spectrum is the feature of different materials, and wherein the described spectrum of at least a portion overlaps at least one of two or more detection windows; And

By the known ratio of the abundant pure sample of each material in described ratio and the different materials relatively being determined the concentration of one or more different materials.

18. method as claimed in claim 17 is characterized in that, described different materials mixes

19. method as claimed in claim 17 is characterized in that, described spectrum is detected in fact simultaneously.

20. method as claimed in claim 17 is characterized in that, described two or more detection windows comprise the detection window of different detectors.

21. method as claimed in claim 17 is characterized in that, described two or more detection windows comprise the different detection windows of a detector.

22. method as claimed in claim 17 is characterized in that, described spectrum has peak value at about identical wavelength place.

23. method as claimed in claim 17 is characterized in that, described spectrum has peak value at the different wave length place.

24. method as claimed in claim 17 is characterized in that, produces described spectrum as the result by the different materials emitting fluorescence.

25. method as claimed in claim 17 is characterized in that, the different continuous part of the whole spectrum of described two or more detection windows span different materials.

26. method as claimed in claim 17 is characterized in that, each in the described output signal has the single value corresponding to the spectral intensity that detects in corresponding detection window.

27. method as claimed in claim 17 is characterized in that, in described two or more detection windows one all is arranged in another of described two or more detection windows.

28. method as claimed in claim 17 is characterized in that, described different materials comprises the material that is associated with microsphere, and described spectrum comprises the different fluorescence emission spectrums of described material.

29. method as claimed in claim 17 is characterized in that, described different materials is included in the material in the solution, and described spectrum comprises difference absorption, transmission or the emission spectrum of described material.

30. method as claimed in claim 17 is characterized in that, described output signal is produced by fluid flow optical device.

31. method as claimed in claim 17 is characterized in that output signal is produced by spectral technique.

32. method as claimed in claim 17 is characterized in that, produces described spectrum as the result by different materials emission, absorption or transmitted light.

33. a computer-implemented method comprises:

Determine that wherein the described spectrum of at least a portion overlaps at least one of two or more detection windows by the ratio between output signals of the spectrum generation of surveying the individual event in two or more detection windows; And

Distinguish between spectrum according to described ratio.

34. a system comprises:

Be configured to survey one or more detectors of the spectrum of the individual event in two or more detection windows, wherein said spectrum is the feature of different materials, wherein the described spectrum of at least a portion overlaps at least one of two or more detection windows, and wherein one or more detectors also are configured to produce the output signal of the spectrum that echo probe arrives; And

Be configured to the processor that any and described ratio in definite ratio between output signals and the definite different materials is associated.

35. system as claimed in claim 34 is characterized in that, described two or more detection windows comprise the detection window of different detectors.

36. system as claimed in claim 34 is characterized in that, described two or more detection windows comprise the different detection windows of a detector.

37. system as claimed in claim 34 is characterized in that, described spectrum has peak value at about identical wavelength place.

38. system as claimed in claim 34 is characterized in that, described spectrum has peak value at the different wave length place.

39. system as claimed in claim 34 is characterized in that, produces described spectrum as the result by the different materials emitting fluorescence.

40. system as claimed in claim 34 is characterized in that, the different continuous part of the whole spectrum of described two or more detection windows span different materials.

41. system as claimed in claim 34 is characterized in that, each in the described output signal has the single value corresponding to the spectral intensity that detects in corresponding detection window.

42. system as claimed in claim 34 is characterized in that, in described two or more detection windows one all is arranged in another of described two or more detection windows.

43. system as claimed in claim 34 is characterized in that, described different materials comprises the material that is associated with microsphere, and described spectrum comprises the different fluorescence emission spectrums of described material.

44. system as claimed in claim 34 is characterized in that, described system is configured to fluid flow optical device and produces.

45. system as claimed in claim 34 is characterized in that, described system is configured to carry out spectral technique.

46. system as claimed in claim 34 is characterized in that, produces described spectrum as the result by different materials emission, absorption or transmitted light.

47. system as claimed in claim 34 is characterized in that, described processor also is configured to the concentration of definite described different materials that is associated with described ratio.

Images