CN1823269A - Method and system for distinguishing between materials having overlapping spectra - Google Patents
Method and system for distinguishing between materials having overlapping spectra Download PDFInfo
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- CN1823269A CN1823269A CN 200480020591 CN200480020591A CN1823269A CN 1823269 A CN1823269 A CN 1823269A CN 200480020591 CN200480020591 CN 200480020591 CN 200480020591 A CN200480020591 A CN 200480020591A CN 1823269 A CN1823269 A CN 1823269A
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Abstract
Various computer-implemented methods and systems are provided. One computer-implemented method includes determining a ratio between output signals generated by detecting spectra for a single event in two or more detection windows. The spectra are characteristic of different materials. At least a portion of the spectra overlap in at least one of the two or more detection windows. The method also includes determining which of the different materials are associated with the ratio. One embodiment of a system includes one or more detectors configured to detect spectra for a single event in two or more detection windows. The spectra may include spectra as described above. The one or more detectors are also configured to generate output signals in response to the detected spectra. The system also includes a processor configured to determine a ratio between the output signals and to determine which of the different materials are associated with the ratio.
Description
Technical field
The present invention relates generally to and is used for the method and system distinguished between the material of overlapping spectra having.Some embodiment relates to and comprises the computer-implemented method of determining which kind of material is associated with the ratio between output signals that produces by the spectrum of surveying the single incident in two or more detecting windows.
Background technology
Spectral technique is widely used in the analysis of chemistry and biosystem.These technology relate to the electromagnetic radiation by interested absorbed and emission mostly.As a rule, the whole relevant portion of the spectrum of studying with low-velocity scanning is to provide absorbing and the measuring the most accurately of emission.Yet, in other systems, only need to check the specific part of spectrum for qualified or definite its quantity of the parameter that proves consideration.For example, if the quantity of sample is many relatively or sample must can be used this inspection by inspection relatively apace.In this case, by reducing the quantity of processed and the raw data analyzed, use " snapshot " of little spectrum can increase the treatment capacity of sample.
A kind of this type of application is in microarray (microarrays) field, this be a kind of by comprise combination chemistry and the technology used of a large amount of subjects of biologicall test industry.Texas Austin's a company, Luminex company limited have developed a kind of system that carries out biologicall test on the microsphere surface of various painted fluorescence.An example of this system has been shown in the 5th, 981, No. 180 United States Patent (USP)s of people such as Chandler.It is quoted just as all being presented and is included in this.When microsphere passed through search coverage with high relatively speed, by laser excitation and fluorescence detection to each independent microsphere, these microspheres were inquired in fluid flow equipment.With several clearly, the detectable signal of each microsphere emission, such system can analyze thousands of microspheres one second.Obviously, handle whole spectrum and the signal from each microsphere in thousands of or millions of microspheres made an explanation and decode to produce unmanageable data volume.Yet the system of being described by people such as Chandler has only realized the management of data by surveying fluorescence in specific " window ", and this window is (for example, about 20nm is to 40nm), the continuous part of lacking relatively from the whole spectrum of microsphere emission.Therefore, not that each microsphere is produced whole fluorescence Spectra, this system only produces single value (intensity of itself and signal is interrelated) to each window.These values can easily output to database and be used for further analysis.
In said system, fluorescent dye is absorbed microsphere into and/or is adhered to the surface of microsphere.Select dyestuff based on ability with the wavelength emission light of the window selected.In addition, a plurality of windows are spaced, and dyestuff is designed to make the overlapping of fluorescence signal in adjacent a plurality of window to minimize and preferably eliminate.By using two windows and two kinds of dyestuffs,, 100 kinds of recognizable microsphere sets of fluorescence will be arranged so separately with 10 kinds of different concentration.
Another example of assay method is shown in the 4th, 717, the 655 good United States Patent (USP)s of Fulwyler, and it is quoted just as all being presented and is included in this.Particularly, Fulwyler has described the method that two or more labelled reagents of a kind of usefulness are distinguished a plurality of subgroups of the cell (cell) that comprises label particles.These particles with multiple different that select in advance and scopes each reagent at the percent 0 ratio marks that arrive the reagent between one of percentage hundred.Each such reagent has significant, quantifiable marker characteristic.In other words, each fluorescent dye has tangible emission and/or excitation spectrum in the color bands of a spectrum of particular design.Mixed mutually with suspecting the cell that the particle of isolabeling is not had a special receptor by the particle of isolabeling not.Analyze the ratio of each cell, if be sorted in the subgroup kind so that the ratio of its marker characteristic ratio and the labelled reagent of preliminary election is relevant with any two discernible marker characteristics of determining to be associated with each cell.Therefore, this method is by detecting the signal from each of two kinds of dyestuffs, utilizes ratio to distinguish by the particle of isolabeling not.
In above-mentioned any system or method, several methods that can expand recognizable group quantity are arranged.Use the microsphere of different size, this microsphere can be distinguished based on light scattering, will the quantity of group be doubled.Another kind method is the quantity that increases every kind of recognizable intensity of dyestuff.For example, if 15 kinds of dye strengths be possible rather than in example 10 in, will obtain 225 groups so.The third method will increase the 3rd window, and be the third dyestuff subsequently, or even more kinds of, this will be with the quantity of index increase group.Different degree has successfully been tested and be used in these methods each.But each increases one deck complicacy to system, and it can greatly increase expense or the difficulty that produces platform.
Summary of the invention
The present invention relates generally to the method for distinguishing between two or more unique but similar spectrums.Some embodiment comprises the signal of detection in two or more different detection windows public to two or all spectrums.The method that is described in this can be used to distinguishing and will find comprising effectiveness in many fields of clinical biological assays between the characteristic population of showing these different spectrums.
An embodiment relates to and comprises the computer-implemented method of determining by the ratio between output signals that in two or more detection windows single incident detection spectrum is produced.As used herein, term " incident " is defined as it is measured with generation and comprises the sample of output signal of significant information or the part of sample.In the environment of clinical biological assays, incident can be a microsphere, particle, or the cell of working as the measurement window of its fluid flow optical device of flowing through (for example, flow cytometers type instrument).Obviously, have can enough terms " incident " many other samples or sample part of describing, and be intended to comprise all possible alternative with the term " incident " here.
Be usually directed to produce the scope of the wavelength or the wavelength of output signal at this place with the term " detection window " here.The wavelength of the irradiation source by being used for shining material and/or the light wavelength that detector can be surveyed can be determined the scope of above-mentioned wavelength or wavelength, and this detector configuration becomes to survey the light by material emission, scattering and transmission.Yet, more generally, the wavelength of detection window will rely on the material distinguished between them and they separately spectrum and change.For example, this spectrum is the characteristic of different materials.In addition, a part of best overlay at least one in two or more detection windows of composing at least.This method also comprises determines that any in the different materials is associated with ratio.In certain embodiments, this method can comprise the concentration of definite different materials that is associated with this ratio.
In one embodiment, the different continuous parts of the whole spectrum of two or more detection windows span different materials.In addition, each in the output signal can have the single value corresponding to the spectral intensity that detects in corresponding detection window.
In one embodiment, two or more detection windows comprise the detection window of different detectors.In an alternative embodiment, two or more detection windows comprise the different detection windows of a detector.In certain embodiments, spectrum has peak value at about identical wavelength place.Perhaps, spectrum has peak value at the different wave length place.In any embodiment, in two or more detection windows can all be arranged in another of two or more detection windows.
Can produce spectrum as result by the light of different materials emission, absorption or transmission.In certain embodiments, produce spectrum as result by different material emitted fluorescence.In another embodiment, different materials comprise the material that is associated with microsphere.In a such embodiment, spectrum can comprise the different fluorescence emission spectrum of this material.In another embodiment, different materials can comprise the multiple material in the solution.In a such embodiment, this spectrum can comprise difference absorption, transmission and the emission of material.In yet another embodiment, spectrum can be included in the combination of the spectrum of two or more materials in the solution.In this embodiment, this method can comprise the independent concentration of determining two or more materials in the solution or ratio.In one embodiment, can produce output signal by fluid flow equipment (for example, flow cytometers type instrument).In other embodiments, can produce output signal by spectral technique.Each embodiment of the method for Miao Shuing can comprise any other step described here in the above.
An additional embodiment relates to another kind of computer implemented method.This method comprises definite ratio between output signals that produces by the spectrum of surveying the individual event in two or more detection windows.This spectrum is the feature of different materials.At least a portion spectrum overlaps at least one of two or more detection windows.This method also comprises by the known ratio of the abundant pure sample of the individual material in ratio and the different materials relatively being determined the concentration of one or more different materials.In one embodiment, different materials is by mixed.In another embodiment, spectrum is surveyed by ground in fact simultaneously.
In one embodiment, two or more detection windows comprise the detection window of different detectors.In different embodiment, two or more detection windows comprise the different detection windows of a detector.The different continuous part of the whole spectrum of two or more detection windows span different materials.In certain embodiments, spectrum has peak value at approximately uniform wavelength place.In other embodiments, spectrum has peak value at different wavelength places.In an additional embodiment, in two or more detection windows one can all be arranged in another of two or more detection windows.
In one embodiment, produced spectrum as result by the different materials emitted fluorescence.Perhaps, the result as different materials emission, absorption or transmission can produce spectrum.In one embodiment, different materials comprises the material that is associated with microsphere, and spectrum comprises the different fluorescence emission spectrums of this material.In another embodiment, different materials can comprise the material in the solution, and spectrum can comprise different absorptions, transmission or the emission spectrum of material.
In certain embodiments, each in the output signal has the single value corresponding to the intensity of the spectrum that detects in corresponding detection window.In one embodiment, produce output signal by fluid flow optical device.In different embodiment, produce output signal by spectral technique.Each embodiment of the method for Miao Shuing can comprise any other step described here in the above.
Another embodiment is relevant with different computer-implemented methods, and this method comprises to be determined by in two or more detection windows single incident being surveyed the ratio between output signals that spectrum produces.At least a portion is composed at least one that overlaps in two or more detection windows.This method is also included between the spectrum according to ratio and distinguishes.This method is also included within any other step of this description.
Method described here is to treat that the two or more different still similar spectrum of differentiating has each other advantageously provided method.Therefore, this method also can distinguished as between the above-mentioned different materials with similar spectrum.Like this, this method has increased the quantity of dye materials, uses method described here to distinguish mutually owing to have the dye materials of similar spectrum, and described dye materials can use in measuring method.According to reading detailed description provided below, the additional advantage of method and system described here will be obvious.
An additional embodiment relates to the system that comprises one or more detectors and processor.One or more detectors are configured to survey the spectrum of the individual event in two or more detection windows.This spectrum is the feature of different materials.At least a portion spectrum overlaps at least one of two or more detection windows.One or more detectors also are configured to the spectrum generation output signal that echo probe is arrived.Processor is configured to determine ratio between output signals.Processor is configured to also to determine that any in the different materials is associated with this ratio.In one embodiment, processor further is configured to the concentration of definite different materials that is associated with this ratio.
The different continuous part of the whole spectrum of two or more detection windows span different materials.In one embodiment, in two or more detection windows can all be arranged in another of two or more detection windows.In addition, each in the output signal can have the single value corresponding to the spectral intensity that detects in corresponding detection window.
In one embodiment, two or more detection windows can comprise the detection window of different detectors.In another embodiment, two or more detection windows comprise the different detection windows of a detector.In certain embodiments, spectrum has peak value at about identical wavelength place.In other embodiments, spectrum has peak value at the different wave length place.
Can produce spectrum as result by the light of different materials emission, absorption or transmission.In one embodiment, produce spectrum as result by different material emitted fluorescence.In another embodiment, different materials comprise the material that is associated with microsphere.In such embodiment, spectrum comprises the different fluorescence emission spectrum of this material.In one embodiment, system is configured to fluid flow equipment.In another embodiment, system is configured to carry out spectral technique.System can further be configured as described here.
Description of drawings
When the reading following detailed description with when with reference to accompanying drawing, other purpose of the present invention and advantage will become more obvious, wherein:
Fig. 1 is the example that the system that is used for carrying out method described here is shown.
Though the present invention can have various modifications and variation, its specific embodiment is illustrated and will describes in detail at this via the example in the accompanying drawing.Yet, be to be understood that, its intention of the drawings and detailed description at this place is not the particular form that invention is limited to announcement, and is on the contrary, is intended to cover all modifications, equivalent and the alternative that belong to by within the spirit and scope of the present invention of additional claims definition.
Embodiment
Notice that a kind of technology that spectrum is described has roughly been described in following explanation.Below explanation will use fluorescence in fluid flow optical device and fluorescent microsphere example application usually as principle.Yet, do not limiting the use of this technology in its intention of this example that provides.For example, it is evident that concerning those skilled in the art that this is a kind of technology that is not limited to fluorescence, particle or fluid flow equipment.Suitable microsphere, bead and particle be the 5th, 736 of Fulton the, No. 330 United States Patent (USP)s, the 5th, 981, No. 180 United States Patent (USP)s of people such as Chandler, the 6th, 057, No. 107 United States Patent (USP)s of Fulton, the 6th, 268 of people such as Chandler, the 222B1 United States Patent (USP), the 6th, 449 of people such as Chandler, the 562B1 United States Patent (USP), the 6th of people such as Chandler, 514,295B1 United States Patent (USP), the 6th of people such as Chandler, 524, the 793B1 United States Patent (USP), the 6th, 528 of people such as Chandler, the 165B2 United States Patent (USP), it is quoted just as all being presented and is included in this.Method described here can be used any microsphere, bead and the particle of describing in these patents.In addition, the microsphere that uses in flow cytometers can obtain from the manufacturer such as Texas Austin's Luminex company limited.
Wherein measurement parameter other spectral technique of being presented at the reappeared distribution in the scope also can use such as infrared ray, ultraviolet ray/visible light (UV/Vis), Raman (Raman), nuclear magnetic resonance (NMR), radioactive emission etc.Equally, detectable parameter can be emission ratio, absorption coefficient, transmission coefficient etc.By any suitable equipment, include but not limited to that photomultiplier, avalanche photodide, charge-coupled image sensor, pin hole diode etc. can be realized the detection of signal.For particle, medium can be solid, liquid, gas in addition, or wherein signal type described here can observed any other form.
In traditional spectroscopy, for example such as fluorescence spectroscopy, the wavelength of source or detector changes in the scope of wavelength to produce the continuous spectrum of checking material.At properties of materials when being known, but its existence or concentration are surveyed in expectation, and a kind of replacement method is to keep exciting and survey the constant and record composite signal of wavelength.This step produces the single value owing to the signal of those specified conditions.Even the shortcoming of this analytical approach is their a full spectrum of analog material is obviously different, also can be in monitored spectrum " window " shows signal.
An example that can be similar signal in spectral window is by rhodamine (Rhodamine) B and rhodamine 6G fluorescent signal emitted.The former has peak emission at 543nm, and the latter has peak emission at 524nm.If detection window is configured to monitor the SPECTRAL REGION from 520nm to 550nm, when being excited by suitable wavelength, these two kinds of signals that dyestuff acquires a special sense demonstration.If known any dyestuff observed, might be determined the concentration of dyestuff in solution based on the signal that is observed.Yet if do not know any dyestuff in system, this signal will not be provided at the method for distinguishing between them.Need more spectral information and make this differentiation.
Use additional detection window can distinguish two kinds of similar spectrums.As mentioned above, the different continuous parts of whole spectrum of detection windows span different materials.Yet,, in the method and system described here, overlapping at least one in two or more detection windows of the spectrum of multiple material unlike the method and system of front.For example, get back in the quoting of example of front, adding the detection window that is configured to survey the spectrum from about 550nm to about 560nm will provide sufficient information distinguishing dyestuff, and can determine concentration thus.Because every kind of dyestuff launches signal widely, each dyestuff with some this signal in this new window.Calculate the ratio R of second window (550nm is to 560nm) signal and first window (520nm is to 550nm) signal, the R that shows rhodamine B is bigger than the R of rhodamine 6G.Importantly, observing the ratio of particular dye will be somewhat constant in the scope of very wide dye strength.The validity of this technology requires the spectrum of dyestuff that enough differences are arranged, and being observed ratio can be distinguished in the working range of concentration consistently.
Therefore, usually,, preferably overlapping at least one in two or more detection windows as at least a portion of the spectrum of the characteristic of different materials.The output signal of detection window can have the single value corresponding to the intensity of the spectrum that detects in corresponding detection window.After this manner, by the spectrum of the individual event of detection in two or more detection windows, and the ratio between output signals that produces can be used for determining that any and this ratio of different materials is associated.Can produce this output signal by fluid flow optical device such as system described here.Perhaps, can produce this output signal by the spectral technique that is included in any this class technology well known in the prior art.
In addition, be important to note that this system is not limited to only distinguish between two kinds of dyestuffs (or other absorption and reflecting material).Quantity by one group of two recognizable dyestuff of window can be expanded together with the wisdom selection to dyestuff by optimizing position of window and width.The ability that produces the unique ratio between the observed signal provides the emission of the spectrum of distinguishing different.Equally, because every kind of dyestuff will produce unique ratio, can be zero from the signal of window to a kind of dyestuff, as long as another kind of dyestuff has in this window than zero bigger signal.Similarly, method described here is not limited to only have two signal detection windows.Although additional window will increase the quantity of processed data, by the ability to see things in their true light of the spectrum using additional window to realize to improve.In addition, the size of the spectrum of detection window can be different from those sizes that provide as an example.For example, the size of detection window is only limited with limited range and the signal that can measure in this scope by the efficient of detection system with reappearing.
Same significant is to notice that two or more spectrums can maybe cannot depart from about their peak (for example peak strength).For example, if the emission spectrum of two kinds of fluorescent dyes shows the peak in about same wave strong point, but the width at a peak is greater than another, and the difference of these in spectrum can cause the unique volume efficiency between the signal in two detection windows so.In these or other embodiment, in two or more detection windows one can or can all not be arranged within another of two or more detection windows.
Further example shows the application of method described here.Under the situation of the microsphere of aforementioned fluorescent dye, by with having the overlapping two kinds of different fluorescent dyes (being appointed as A and B here) of minimum spectrum can produce microsphere to group's dyeing of microsphere 100 kinds of groups that spectrum is different.This type of example is shown in the 6th, 514, No. 295 United States Patent (USP)s of people such as Chandler, and it is quoted just as all being presented and is included in this.If two kinds of solution can produce 100 kinds of different staining solutions and 100 kinds of microsphere groups that fluorescence is different so with 10 kinds of variable concentrations preparations in two kinds of dyestuffs each.Use can be inquired the flow cytometers or the miscellaneous equipment of independent microsphere or microsphere sets with spectrum, can use these unique microspheres of spectrum identification.
Detection transmits by (for example, in two windows, be appointed as window 1 and window 2 at this) in narrow wavelength bands of a spectrum window, can use the spectrum identification microsphere.In addition, window 1 is selected to the signal of surveying effectively from dyestuff A, and window B is selected to corresponding to the emission from dyestuff B.If added the 3rd window (being window 3), it near window 2, can select also to launch the third dyestuff (dyestuff B ') by window 2 and window 3 detectable signals so on spectrum, and causes the unique ratio between window 2 and the window 3.Then, set up 100 kinds of new dye solutions, can set up 100 kinds of new fluorescent microsphere groups by utilizing each the 10 kinds of variable concentrations among dyestuff A and the B '.Can find out easily that adding can produce the another kind of dyestuff of unique ratio (dyestuff B ") between from window 2 and window 3, uses dyestuff A and B " will constitute 100 kinds of extra unique bead groups.Extra group energy produces enough in a similar manner, as long as can find to produce the dyestuff of unique ratio.In addition, if add another window (window 4), it is close window 1 on spectrum, then use new dyestuff (dyestuff A ', A " etc.) and dyestuff B, B '; B " can constitute similar series Deng combination, greatly to enlarge the potential quantity of unique fluorescent microsphere sets.
In another embodiment, this method can be used for the overlapped spectra that is determined is in fact simultaneously removed to revolve (deconvolute), can see now such as mixed being at compound.For example, the material that invests on the microsphere can be distinguished as mentioned above.In such embodiment, this method can comprise by the ratio and the known ratio of the abundant pure sample of the independent material that is constituted different materials by conjecture relatively being determined the concentration of one or more different materials.Yet, use any other method of the prior art, this concentration can be determined according to ratio.After this manner, when the dye strength of determining was used to determine the microsphere identity, this method can be used to use many information (multi-reporter) of two or more dyestuffs to discern microsphere in measuring at spherome surface.Such material also can comprise, for example, is attached to the fluorescer of nucleic acid, enzyme, antigen etc. in some way.Another example is the solution of two or more dyestuffs, and it is supposed to be used for detecting or determining the concentration or the concentration rate of every kind of dyestuff.Particularly, spectrum can be included in the spectrum of different materials in the solution.In such an embodiment, this spectrum can comprise difference absorption, transmission and the emission spectrum of this material.In addition, spectrum can be included in the combination of the spectrum of two or more materials in the solution.This method also can comprise determines independent concentration or ratio to two or more materials in the solution.
In aforesaid such example, use is as the fluorescent dye of an example, if dyestuff A is by self being created in two ratios 1 between the detection window, and dyestuff B produces ratio 100 by self, so the mixed ratio that will be created between 1 to 100 of two kinds of dyestuffs.In addition, if potpourri mainly is made up of dyestuff A, then observed ratio will be more near 1; And if when more dyestuff B was arranged, this ratio will be more near 100.Therefore, this ratio will be relevant with the component of dye mixture, so that for specific ratio, might calculate the ratio of forming the observed signal that is caused owing to every kind of dyestuff.Therefore in the part of this signal that is attributable to every kind of dyestuff when being known, and signal is known to the correlativity of dye strength, and the concentration of two kinds of dyestuffs can be determined simultaneously so.May cause new problem by interacting between the dyestuff such as cancellation (quenching) and other energy transfer phenomenon, can be resolved but these problems make up typical curve by the combination of using known dye strength.The complicacy of analyzing the spectrum of three kinds or multiple overlapping dye mixture may increase significantly, may produce several potential solutions.Yet, might remove incorrect scheme by other means.
Fig. 1 shows an example that can be used on the measuring system of carrying out method described here.Notice that Fig. 1 does not press ratio and draws.Particularly, the ratio of some element of figure is greatly amplified to emphasize the feature of element.
In Fig. 1, show measuring system along the plane of the xsect of the test tube 12 of flowing through by microsphere 10.In one embodiment, test tube can be such as the standard quartz test tube that uses in the flow cytometers of standard.Yet the observation of any other suitable type or transfer chamber also can be used to transmit the sample that is used to analyze.This measuring system comprises light source 14.Light source 14 can comprise the well known in the prior art any suitable light source such as laser.Light source 14 can be configured to launch the light with one or more wavelength such as blue light or green glow.Light source 14 can be configured to it be shone during through test tube when microsphere flow.This irradiation can make the microsphere emission have the fluorescence of one or more wavelength or wavelength bands of a spectrum.In certain embodiments, this system can comprise and is configured to the light from light source is focused on one or more lens (not shown) on microsphere or the flow process.This system also can comprise more than one light source.In one embodiment, light source can be configured to light (for example, blue light and green glow) the irradiation microsphere with different wave length.In certain embodiments, light source can be configured to different direction irradiation microspheres.
Can sensing system 16 from the light of microsphere forescatering by folding mirror or another kind of light directional component.Perhaps, detection system 16 can directly be placed in the light path of forescatering.Like this, folding mirror or other photoconduction can be not included in this system to assembly.In one embodiment, as shown in fig. 1, the light of forescatering can be with the light from the about 180 degree angle scatterings of the direction of light source 14 irradiations by microsphere.The angle of the light of forescatering can not be from the direction of light source 14 irradiation 180 degree just in time, so that be not incident on the photosensitive surface of detection system from the incident light of light source.For example, the light of forescatering can be by microsphere with from the direction of light source 14 irradiation less than or greater than the light of the scattered through angles of 180 degree the light of about 170 degree, about 175 degree, about 185 degree or about 190 degree angle scatterings (for example).
By microsphere also can be collected from the about an angle of 90 degrees scattering of direction of light source 14 irradiation and/or the light of emission.In one embodiment, this scattered light can be divided into more than one light beam by one or more spectroscopes or dichronic mirror.For example, can be divided into two with light by spectroscope 20 and restraint different light beams from the about an angle of 90 degrees scattering of direction of light source 14 irradiation.The different light beam of this two bundle is separated again to produce the different light beam of four bundles by spectroscope 22 and 24.Every light beams can the different detection system of directive, and detection system can comprise one or more detectors.For example, a branch of in four light beams can directive detection system 26.Detection system 26 can be configured to survey the light by the microsphere scattering.
But other three-beam directive detection system 28,30 and 32. Detection system 28,30 and 32 can be configured to survey the fluorescence of being launched by microsphere.Each detection system can be configured to survey the fluorescence of different wave length or different wavelength range.For example, a kind of detection system can be configured to survey green fluorescence.Another kind of detection system is configured to survey orange fluorescence.Also a kind of detection system is configured to survey red fluorescence.In another embodiment, different detectors has different detection windows, as above in the further described detection window at least one, at least a portion of the spectrum of different materials is overlapping.In a different embodiment, one in the detector can have different detection windows, and in one in detection window, at least a portion of the spectrum of different materials is overlapping.An example that can have the detector of a plurality of detection windows is multi-anode photomultipliers, and wherein each anode can be used as different detection windows.
In certain embodiments, spectral filter 34,36 and 38 can be coupled to detection system 28,30 and 32 respectively.Spectral filter can be configured to stop the fluorescence those wavelength that are configured to survey except that detection system.In addition, one or more lens (not shown) can be coupled to each detection system optically.Lens can be configured to scattered light or emitted fluorescence are focused on the photosensitive surface of detector.
The output current of detector is proportional to the fluorescence that is incident on above it and causes current impulse.Current impulse is convertible into potential pulse, and low-pass filtering is carried out digitizing by the A/D converter (not shown) then.The numerical value of quadraturing and representing the fluorescence amplitude such as the zone under processor 40 paired pulses of DSP to provide.In addition, processor can be carried out other function described here (for example, determining that any of ratio between output signals and definite different materials is associated with this ratio).Shown in Fig. 1, processor 40 can be coupled to detector 26 via transmission medium 42.Processor 40 also can be coupled to detector 26 indirectly via transmission medium 42 with such as one or more other parts (not shown) of A/D converter.Processor can be coupled to other parts of system with similar mode.
In certain embodiments, can be processed according to the output signal that produces by the microsphere emitted fluorescence with the identity of definite microsphere and the information of the relevant reaction that takes place on the surface of microsphere.For example, can be used to determine that from the output signal of two or more detectors the identity of microsphere, other output signal can be used to determine the reaction that takes place on the microsphere surface.Can determine the identity of microsphere based on the ratio of the output signal that in two or more different windows, produces.For example, if detection system 30 and 32 has different detection windows, to the ratio of the output signal that produces by detection system 32 intensity, can determine the identity of microsphere together with each signal according to the output signal that produces by detection system 30.Therefore, the selection of detector and spectral filter relies on the type (that is, being included in or being adhered to the dyestuff of the reactant that relates to this reaction) of the dyestuff be included in or be adhered to microsphere and/or measured reaction and difference.
In a certain embodiments, the selection of detector and/or spectral filter can rely on the peak value of the dyestuff that is included in or is adhered to microsphere.For example, as mentioned above, rhodamine B has the peak emission at 543nm, and rhodamine 6G has the peak emission at 524nm.If microsphere is dyeed with different concentration with a kind of or these two kinds of dyestuffs, detection system 30 can be configured to survey light in the wavelength coverage from about 520nm to about 550nm.In addition, detection system 32 can be configured to survey light in the wavelength coverage from about 550nm to about 560nm.
In another embodiment, as mentioned above, the emission spectrum of two kinds of fluorescent dyes cannot depart from, but can show peak emissions at about identical wavelength place.In such example, can be different in the characteristic of the either side of peak emission or the emission spectrum on the both sides.Similarly, emission spectrum has in two detection windows unique volume efficiency between the signal.Therefore, in one embodiment, although detection system 30 and 32 has different detection windows, one detection window in the detection system can all or part ofly be positioned at the detection window of another detection system.For example, if the peak emission of two kinds of dyestuffs at about 540nm, then the detection window of detection system 30 can have the wavelength coverage from about 530nm to about 550nm, the detection window of detection system 32 can have the wavelength coverage from about 510nm to about 570nm.Wavelength coverage above noting only is an example and according to the dyestuff of for example microsphere and difference.
Although being illustrated, the system of Fig. 1 comprises detection system with two different detection windows that are used between microsphere, distinguishing with different dyes characteristic, the system of being to be understood that can comprise more than such detection window of two (promptly, 3 detection windows, 4 detection windows etc.).In such embodiments, system can comprise additional spectroscope and the additional detection system with other detection window.Be used for to be determined as mentioned above more than the detection window of two detection systems.In addition, spectral filter or lens can be coupled to each additional detection system.
In another embodiment, system can comprise be configured to two or more detection systems of distinguishing between the different materials that reacts on the surface of microsphere.Different reactant materials can have the dye characteristic different with the dye characteristic of microsphere.Yet reactant materials can have such dye characteristic, so that they have similar emission spectrum.For example, the emission spectrum of reactant materials can be overlapping.In one embodiment, emission spectrum can have offset peak emissions, but also can show strong signal at one or more identical wavelength places.In a different embodiment, emission spectrum is on the either side or both sides of peak emission, and but emission spectrum can have roughly the same peak emission different features.Therefore, emission spectrum and reactant materials can be distinguished as mentioned above.
Can be used to carry out the additional examples of the measuring system of method described here, the 5th, 981 of people such as Chandler, No. 180 United States Patent (USP)s, the 6th, 046 of Chandler, No. 867 United States Patent (USP)s, the 6th, 139 of Chandler, No. 800 United States Patent (USP)s, the 6th, 366, No. 354 United States Patent (USP)s of Chandler, the 6th of Chandler, 411, No. 904 United States Patent (USP)s, the 6th of people such as Chandler, 449, No. 562 United States Patent (USP)s, the 6th, 524 of people such as Chandler, shown in No. 793 United States Patent (USP)s, it is quoted just as all being presented and is included in this.The system of describing in these patents can be configured to have aforesaid system.Measuring system described here also can be further configured as described in these patents.
Can in mounting medium, send or be stored in the mounting medium such as those programmed instruction implementation methods described here.Mounting medium can be the transmission medium such as lead, cable or wireless transmission link, or the signal of propagating along such lead, cable or circuit.Mounting medium also can be the storage medium such as ROM (read-only memory), random access memory, disk or CD or tape.
In one embodiment, can be configured to the method implemented with object computer according to top embodiment execution of program instructions such as the processor of the processor 40 of Fig. 1.This processor can adopt multi-form, comprises DSP, personal computer system, host computer system, workstation, the network equipment, internet equipment, personal digital assistant (" PDA "), television system or miscellaneous equipment.Usually, term " computer system " can broadly be defined as and comprise any equipment with one or more processors, and processor can be carried out the instruction from storage medium.
Programmed instruction can be performed with any different mode, comprising technology based on step, and based on the technology of parts, and/or OO technology.For example, use ActiveX control, C++ object, JavaBeans, microsoft foundation class (" MFC ") or other technology or method, can be as expectation execution of program instructions.
Should be appreciated that concerning those open thus those skilled in the art that are benefited that the present invention aims to provide is used for the method distinguished between similar absorption, transmission and emission spectrum.According to this instructions, the modification of each side of the present invention and alternative embodiment will be conspicuous to those skilled in the art.Therefore, this instructions only is interpreted as exemplary and purpose is to be used for teaching those skilled in the art in the ordinary way to carry out the present invention.Should be understood that form of the present invention shown and described herein is considered to as present preferred embodiment.To can be replaced at shown in this and element and material that describe, parts and process can be changed, some feature of the present invention can be utilized separately, and all these are conspicuous for a technician in this area after instructions of the present invention is benefited.Under the spirit and scope of the present invention of in not deviating from, describing, can in element described here, make a change as claims in the back.
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.
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| US48860003P | 2003-07-18 | 2003-07-18 | |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419159B (en) * | 2008-11-24 | 2011-09-28 | 杨季冬 | Spectral analysis method for simultaneous determination of spectrum peak complete-superposing two-component mixture |
| CN104813160A (en) * | 2012-06-01 | 2015-07-29 | 威瑞泰得有限公司 | Real time optical detection of bacteria |
| CN106680186A (en) * | 2016-11-02 | 2017-05-17 | 北京信息科技大学 | Multi-type scattered light detection system of flow cytometer |
-
2004
- 2004-07-07 CN CN200480020591A patent/CN100587472C/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419159B (en) * | 2008-11-24 | 2011-09-28 | 杨季冬 | Spectral analysis method for simultaneous determination of spectrum peak complete-superposing two-component mixture |
| CN104813160A (en) * | 2012-06-01 | 2015-07-29 | 威瑞泰得有限公司 | Real time optical detection of bacteria |
| US9939378B2 (en) | 2012-06-01 | 2018-04-10 | Veritide Limited | Real time optical detection of bacteria |
| CN106680186A (en) * | 2016-11-02 | 2017-05-17 | 北京信息科技大学 | Multi-type scattered light detection system of flow cytometer |
| CN106680186B (en) * | 2016-11-02 | 2019-03-01 | 北京信息科技大学 | A kind of flow cytometer polymorphic type scattering optical detection system |
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| CN100587472C (en) | 2010-02-03 |
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