Abstract: A fluorescence detection system, including apparatus and methods, suitable for qPCR and other fluorescence-based analyses. The system may comprise various components, including a stage, an illumination module, a detection module, and an optical relay structure. The stage may be configured to support a sample holder. The illumination module may include one or more discrete light sources configured to produce excitation light. The detection module may be configured to detect fluorescence emission light produced, in response to the excitation light, by a fluorescent sample positioned in the sample holder. The optical relay structure may include a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the imaging module. The system may enhance the quality of excitation light hitting samples in the sample holder.

Abstract: A method of analysis for alleles of a target includes forming plural fluid volumes. Each of those volumes includes first and second primer pairs to amplify corresponding first and second alleles of a target. Each of those volumes also includes first and second reporters with corresponding first and second photoluminophores and providing corresponding primers of the corresponding first and second primer pairs. Each of the first and second reporters may include an oligomer that has a quencher and is configured to base-pair with the primer of the corresponding first and second primer pair. The first and second alleles may be amplified using the corresponding first and second primer pairs. The method detects photoluminescence and determines a level of each allele.

Abstract: Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.

Abstract: A fluorescence detection system, including apparatus and methods, suitable for qPCR and other fluorescence-based analyses. The system may comprise various components, including a stage, an illumination module, a detection module, and an optical relay structure. The stage may be configured to support a sample holder. The illumination module may include one or more discrete light sources configured to produce excitation light. The detection module may be configured to detect fluorescence emission light produced, in response to the excitation light, by a fluorescent sample positioned in the sample holder. The optical relay structure may include a beamsplitter assembly configured to direct the excitation light from the illumination module along an illumination path to the sample holder and to direct the fluorescence emission light from the sample holder along a response path to the imaging module. The system may enhance the quality of excitation light hitting samples in the sample holder.

Abstract: Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: Methods of, and compositions for, analysis using digital amplification assays with unconventional/inverse changes in photoluminescence to indicate the presence of one or more targets. In an exemplary method, isolated volumes may be formed, only a subset of which contain a target. Each volume may include a probe having a label, a sink having a quencher and configured to hybridize with the probe to quench the label, and a separator configured to hybridize with the probe and/or the sink to block hybridization of the sink with the probe. An amplification reaction may be performed in the volumes to generate an amplicon corresponding to the target. The separator may hybridize with the amplicon, and may be extended or degraded by the amplification reaction. Photoluminescence of the label may be detected from the volumes, and the photoluminescence of target-positive volumes may be less than that of target-negative volumes.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: Methods of, and compositions for, analysis using digital amplification assays with unconventional/inverse changes in photoluminescence to indicate the presence of one or more targets. In an exemplary method, isolated volumes may be formed, only a subset of which contain a target. Each volume may include a probe having a label, a sink having a quencher and configured to hybridize with the probe to quench the label, and a separator configured to hybridize with the probe and/or the sink to block hybridization of the sink with the probe. An amplification reaction may be performed in the volumes to generate an amplicon corresponding to the target. The separator may hybridize with the amplicon, and may be extended or degraded by the amplification reaction. Photoluminescence of the label may be detected from the volumes, and the photoluminescence of target-positive volumes may be less than that of target-negative volumes.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: An automated system captures multiple digital images of a sample, from different viewing areas of the sample. The images are analyzed and a test result may be reported based on the count of cells in all of the digital images. The system may be especially applicable to the testing of samples having low cell concentrations, as the variability between tests may be reduced as compared with a system that bases its test results on only a single image of the sample. The system may count the cells in a first digital image of the sample, and may capture or analyze one or more additional digital images only when it is recognized that additional measurements are needed to ensure adequate accuracy and repeatability of the test.

Abstract: System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Abstract: An automated system captures multiple digital images of a sample, from different viewing areas of the sample. The images are analyzed and a test result may be reported based on the count of cells in all of the digital images. The system may be especially applicable to the testing of samples having low cell concentrations, as the variability between tests may be reduced as compared with a system that bases its test results on only a single image of the sample. The system may count the cells in a first digital image of the sample, and may capture or analyze one or more additional digital images only when it is recognized that additional measurements are needed to ensure adequate accuracy and repeatability of the test.