Apparatus and method for differentiating multiple fluorescence signals by excitation wavelength
First Claim
1. An apparatus comprising:
- a thermal cycler;
a plurality of reaction regions, each comprising a respective sample disposed therein, the plurality of reaction regions being connected to the thermal cycler, the thermal cycler being adapted to carry out thermal cycling to effect a polymerase chain reaction on the respective samples in the plurality of reaction regions;
a plurality of detectors disposed above the samples and capable of detecting emission beams emitted from the plurality of reaction regions, the plurality of detectors including a different detector centered above each one of the reaction regions;
for each detector, four or more excitation light sources capable of providing a plurality of separate excitation beams, each excitation beam comprising a wavelength range that is not provided by the other excitation beams of the plurality of separate excitation beams, each excitation light source being capable of directing a respective excitation beam along a beam path toward the respective reaction region, the four or more excitation light sources for each detector surrounding the respective detector and being spaced above the respective sample disposed in the reaction region;
a control unit capable of activating each of the four or more excitation light sources for each detector independently from the other excitation light sources of the respective four or more excitation light sources; and
a detection system configured to control the plurality of detectors to simultaneously detect emission beams from each of the plurality of reaction regions and to associate the emission beams detected from each of the plurality of reaction regions with the excitation light source activated by the control unit.
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Abstract
An apparatus and method are provided for differentiating multiple detectable signals by excitation wavelength. The apparatus can include a light source that can emit respective excitation light wavelengths or wavelength ranges towards a sample in a sample retaining region, for example, in a well. The sample can contain two or more detectable markers, for example, fluorescent dyes, each of which can be capable of generating increased detectable emissions when excited in the presence of a target component. The detectable markers can have excitation wavelength ranges and/or emission wavelength ranges that overlap with the ranges of the other detectable markers. A detector can be arranged for detecting an emission wavelength or wavelength range emitted from a first marker within the overlapping wavelength range of at least one of the other markers.
166 Citations
63 Claims
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1. An apparatus comprising:
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a thermal cycler; a plurality of reaction regions, each comprising a respective sample disposed therein, the plurality of reaction regions being connected to the thermal cycler, the thermal cycler being adapted to carry out thermal cycling to effect a polymerase chain reaction on the respective samples in the plurality of reaction regions; a plurality of detectors disposed above the samples and capable of detecting emission beams emitted from the plurality of reaction regions, the plurality of detectors including a different detector centered above each one of the reaction regions; for each detector, four or more excitation light sources capable of providing a plurality of separate excitation beams, each excitation beam comprising a wavelength range that is not provided by the other excitation beams of the plurality of separate excitation beams, each excitation light source being capable of directing a respective excitation beam along a beam path toward the respective reaction region, the four or more excitation light sources for each detector surrounding the respective detector and being spaced above the respective sample disposed in the reaction region; a control unit capable of activating each of the four or more excitation light sources for each detector independently from the other excitation light sources of the respective four or more excitation light sources; and a detection system configured to control the plurality of detectors to simultaneously detect emission beams from each of the plurality of reaction regions and to associate the emission beams detected from each of the plurality of reaction regions with the excitation light source activated by the control unit. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method comprising:
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providing a set of samples that includes at least two detectable markers, wherein each detectable marker emits a respective detectable emission beam when illuminated by an excitation beam that includes light of a respective wavelength; thermally cycling the set of samples to perform a polymerase chain reaction; illuminating the set of samples with a first excitation beam, directed from a first excitation light source disposed above the samples, that includes a first excitation wavelength, during the thermal cycling; simultaneously detecting first emission beams emitted from the set of samples, with a plurality of detectors including a different detector centered above each one of the samples, in response to illuminating the set of samples with the first excitation beam, during the thermal cycling, wherein the first emission beams include a first emission wavelength emitted from at least one sample of the set of samples; independently illuminating the set of samples with a second excitation beam, that differs from the first excitation beam, directed from a second excitation light source disposed above the samples, that includes a second excitation wavelength that differs from the first excitation wavelength, and that does not include the first excitation wavelength, during the thermal cycling; and simultaneously detecting second emission beams emitted from the set of samples with the plurality of detectors in response to illuminating the set of samples with the second excitation beam, during the thermal cycling, wherein the second emission beams include the first emission wavelength emitted from at least one sample of the set of samples. - View Dependent Claims (27, 28, 29)
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30. A method of differentiating multiple fluorescent signals by excitation wavelength, comprising:
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(a) providing a respective sample in each of a plurality of sample wells, each sample comprising a plurality of components; (b) adding four or more fluorescent dyes to each sample, each of the four or more fluorescent dyes being capable of generating increased emission due to a presence of a respective target component in the sample, each of the four or more fluorescent dyes possessing a respective excitation wavelength range and a respective emission wavelength range; (c) thermally cycling each sample to perform a polymerase chain reaction; (d) directing beams of at least four different excitation wavelength ranges from a different light source disposed above each respective sample toward the sample to cause at least a respective four of the fluorescent dyes to emit a detectable emission, during the thermal cycling; (e) simultaneously detecting emission beams emitted from each sample with a plurality of detectors including a different detector centered above each respective sample, during the thermal cycling; and (f) determining whether each of four respective target components is present in the sample by analyzing the detected emission wavelengths. - View Dependent Claims (31, 32, 33, 34, 35)
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36. An analytical instrument comprising:
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a thermal cycler; a plurality of reaction regions, each comprising a respective sample disposed therein, the plurality of reaction regions being connected to the thermal cycler, the thermal cycler being adapted to carry out thermal cycling to effect a polymerase chain reaction on the respective samples in the plurality of reaction regions; at least one excitation beam source array comprising a plurality of light sources comprising four or more excitation beam source portions and including a different light source disposed above each respective one of the plurality of samples, the four or more excitation beam source portions being controllable to emit at least four different excitation wavelengths; a control unit capable of activating each of the four or more excitation beam source portions independently from the others of the four or more excitation beam source portions; at least one detector array comprising a plurality of detector portions including a different detector portion spaced above and centered above each respective one of the samples; and a detector system configured to control the detector array to simultaneously detect emission beams from each of the plurality of reaction regions and to associate the emission beams detected from each of the plurality of detection regions with the excitation beam source portion activated by the control unit; wherein at least one of the plurality of the excitation beam source portions is arranged between two of the plurality of detector portions, at least one of the plurality of detector portions is arranged between two of the plurality of excitation beam source portions, and each of the at least four different excitation wavelengths emitted from the four or more excitations beam source portions is divided to provide excitation beams to two or more of the plurality of reaction regions. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. A system comprising:
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an analytical instrument including a plurality of reaction regions, each region comprising a respective sample disposed therein, the plurality of reaction regions being connected to a thermal cycler, the thermal cycler being adapted to carry out thermal cycling to effect a polymerase chain reaction on the respective samples in the plurality of reaction regions; at least one excitation beam source array comprising a plurality of light sources each comprising four or more excitation beam source portions, a different light source being disposed above each of the respective samples, the four or more excitation beam source portions being controllable to emit at least four different excitation on wavelengths; at least one detector array comprising a plurality of detector portions including a different detector portion disposed above each of the respective samples; a detector system configured to control the detector array to simultaneously detect emission beams from each of the plurality of reaction regions, wherein at least one of the four or more excitation beam source portions or at least one of the plurality of detector portions is arranged and centered between two of the other; and a sample-containment component including a plurality of sample-containment units, wherein each of the four or more excitation beam source portions is operatively aligned to direct excitation beams towards a different one of the sample-containment units, and each of the plurality of detector portions is centered above a different one of the plurality of sample-containment units. - View Dependent Claims (48, 49, 50, 51, 52, 53)
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54. A method of irradiating samples in sample-containment units, the method comprising:
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providing a thermal cycler; providing an analytical instrument comprising a plurality of reaction regions, each reaction region comprising a respective sample disposed therein, the plurality of reaction regions being connected to the thermal cycler, the thermal cycler being adapted to carry out thermal cycling to effect a polymerase chain reaction on the respective samples in the plurality of reaction regions, at least one excitation beam source array comprising a plurality of light sources, each light source comprising four or more excitation beam source portions and being disposed above a different one of the respective samples, at least one detector array including a plurality of detector portions including a different detector portion disposed above each of the respective samples in the plurality of reaction regions, and a detector system configured to control the detector array to simultaneously detect emission beams from each of the plurality of reaction regions, wherein at least one of the four or more excitation beam source portions or at least one of the plurality of detector portions is arranged and centered between two of the other; thermally cycling the respective samples to effect a polymerase chain reaction; generating at least four excitation beams having different excitation wavelengths with at least four of the four or more excitation beam source portions, during the thermal cycling; and directing the at least four excitation beams toward the respective samples. - View Dependent Claims (55, 56, 57, 58, 59, 60, 61, 62, 63)
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Specification