The present application provides for systems and methods for detecting and estimating signals corresponding to one or more biomarkers in biological samples stained for the presence of protein and/or nucleic acid biomarkers. On particular aspect is directed to a method of estimating an amount of signal corresponding to at least one biomarker in an image of a biological sample. The method includes detecting isolated spots in a first image, deriving an optical density value of a representative isolated spot based on signal features from the detected isolated spots, estimating a number of predictive spots in signal aggregates in each of a plurality of sub-regions based on the derived optical density value of the representative isolated spot, and storing the estimated number of predictive spots and detected isolated spots in each of the plurality of generated sub-regions in a database.

The present application provides for systems and methods for detecting and estimating signals corresponding to one or more biomarkers in biological samples stained for the presence of protein and/or nucleic acid biomarkers. On particular aspect is directed to a method of estimating an amount of signal corresponding to at least one biomarker in an image of a biological sample. The method includes detecting isolated spots in a first image, deriving an optical density value of a representative isolated spot based on signal features from the detected isolated spots, estimating a number of predictive spots in signal aggregates in each of a plurality of sub-regions based on the derived optical density value of the representative isolated spot, and storing the estimated number of predictive spots and detected isolated spots in each of the plurality of generated sub-regions in a database.

Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleotide connected to a nucleic acid comprising a FRET label comprising at least three luminescent molecules. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided.

Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleotide connected to a nucleic acid comprising a FRET label comprising at least three luminescent molecules. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

An apparatus for characterizing luminescent entities by excitation comprising: • a substrate (6) being in contact with a solution comprising luminescent entities; • a source of electromagnetic radiation (4) providing at least a primary beam of radiation (8); an objective (5); a first optical element (1) capable of transforming the intensity profile of the primary beam (8) into an arbitrary secondary intensity profile (distribution) (9); a second optical element (2) capable of separating (discriminating) radiation by wavelength; and a detector (7), where the arbitrary secondary intensity profile has at least an off-center circular continuous intensity distribution (33) focused on the back focal plane (12) of the objective forming a collimated beam (10) capable of creating an evanescent field on the side of the substrate where the solution comprising luminescent entities are located, where the evanescent field excites the luminescent entities thereby creating emission radiation separated by the second optical element (2) and captioned by the detector (7). The invention also relates to an apparatus comprising two optical elements providing a final third intensity profile (distribution) which is the convolution of two mathematical transformations corresponding to each of optical element one and four, respectively.

A detection method including bringing liquid including a target nucleic acid into contact with a well array having wells such that at most one molecule of the target nucleic acid is included per well, sealing the well such that the target nucleic acid remains in the well, amplifying, in the well, a signal derived from the target nucleic acid, detecting the signal emitted from the well, and detecting whether the target nucleic acid includes at least one of a first nucleic acid, a second nucleic acid, and a double-stranded nucleic acid resulting from complementary binding of the first and second nucleic acids. The signal includes at least one of a first signal emitted by binding of a first specific binding substance to the first nucleic acid, and a second signal different from the first signal and emitted by binding of a second specific binding substance to the second nucleic acid.

A detection method including bringing liquid including a target nucleic acid into contact with a well array having wells such that at most one molecule of the target nucleic acid is included per well, sealing the well such that the target nucleic acid remains in the well, amplifying, in the well, a signal derived from the target nucleic acid, detecting the signal emitted from the well, and detecting whether the target nucleic acid includes at least one of a first nucleic acid, a second nucleic acid, and a double-stranded nucleic acid resulting from complementary binding of the first and second nucleic acids. The signal includes at least one of a first signal emitted by binding of a first specific binding substance to the first nucleic acid, and a second signal different from the first signal and emitted by binding of a second specific binding substance to the second nucleic acid.

Embodiments disclosed herein include devices, methods, and systems for direct, selective, and sensitive detection of single-stranded and double-stranded target DNA sequences from various sources using a Cas12a protein. When activated by binding a target DNA sequence, the Cas12a cleaves a tether releasing a reporter molecule that may then be detected. In some embodiments, the systems, methods, and devices may include a filter or membrane that may help to separate the tethered and untethered reporter molecules. These devices, systems, and techniques allow a user to rapidly process samples that may contain the target DNA, without needing to amplify the target sequences. These devices and methods may be used to assay a wide variety of samples and target DNA sources, for the presence or absence of target DNA sequences. Compositions and kits, useful in practicing these methods, for example detecting a target DNA in a biological sample, are also described.

Embodiments disclosed herein include devices, methods, and systems for direct, selective, and sensitive detection of single-stranded and double-stranded target DNA sequences from various sources using a Cas12a protein. When activated by binding a target DNA sequence, the Cas12a cleaves a tether releasing a reporter molecule that may then be detected. In some embodiments, the systems, methods, and devices may include a filter or membrane that may help to separate the tethered and untethered reporter molecules. These devices, systems, and techniques allow a user to rapidly process samples that may contain the target DNA, without needing to amplify the target sequences. These devices and methods may be used to assay a wide variety of samples and target DNA sources, for the presence or absence of target DNA sequences. Compositions and kits, useful in practicing these methods, for example detecting a target DNA in a biological sample, are also described.