This invention relates to systems and methods for imaging sample materials within a microfluidic device during an assay reaction process. In accordance with certain aspects of the invention, images are formed with a pixel array and a region of interest (ROI) is defined within the pixel array. Image values, such as fluorescent intensity, can be computed as averages of individual pixel values within the ROI. Where the ROI is subject to non-uniform conditions, such as non-uniform heating, the ROI can be divided into sub-ROIs which are sufficiently small that the condition is uniform within the sub-ROI.

A method for determining a presence or absence of one or more target analytes in a sample includes contacting the sample with an array of particles comprising at least first and second particle subsets disposed therein with a known particle number ratio with respect to each other. The first particle subset has at least one binding site configured to bind with a first target analyte and the second particle subset has at least one binding site configured to bind with a second, different target analyte. Changes are detected in a detectable signal emitted by the particles after contacting the sample with the array. A number of the particles that emit the change in the detectable signal are counted and this number is compared to the known particle number ratio of the subsets so as to determine the presence or absence of the one or more of the target analytes.

A method for determining a presence or absence of one or more target analytes in a sample includes contacting the sample with an array of particles comprising at least first and second particle subsets disposed therein with a known particle number ratio with respect to each other. The first particle subset has at least one binding site configured to bind with a first target analyte and the second particle subset has at least one binding site configured to bind with a second, different target analyte. Changes are detected in a detectable signal emitted by the particles after contacting the sample with the array. A number of the particles that emit the change in the detectable signal are counted and this number is compared to the known particle number ratio of the subsets so as to determine the presence or absence of the one or more of the target analytes.

Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.

Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.

A method for detecting an analyte in a sample includes the steps of binding a first catalytic precursor to the analyte at a first epitope and binding a second catalytic precursor to the analyte at a second, different epitope to generate a catalytic complex. The catalytic complex is reacted with a multiplex molecular substrate to generate a first target molecule and an intermediate substrate containing the bound catalytic complex. The intermediate substrate is reacted with a dummy reactant to generate a second target molecule, wherein the reaction further generates a waste molecule containing the dummy reactant and a free catalytic complex. An optical signal that is generated by one or more dye(s) specific to the first target molecule and/or the second target molecule is detected to detect the presence of the analyte in the sample. The overall reaction has substantially net zero enthalpy and a positive entropy change.

A method for detecting an analyte in a sample includes the steps of binding a first catalytic precursor to the analyte at a first epitope and binding a second catalytic precursor to the analyte at a second, different epitope to generate a catalytic complex. The catalytic complex is reacted with a multiplex molecular substrate to generate a first target molecule and an intermediate substrate containing the bound catalytic complex. The intermediate substrate is reacted with a dummy reactant to generate a second target molecule, wherein the reaction further generates a waste molecule containing the dummy reactant and a free catalytic complex. An optical signal that is generated by one or more dye(s) specific to the first target molecule and/or the second target molecule is detected to detect the presence of the analyte in the sample. The overall reaction has substantially net zero enthalpy and a positive entropy change.

The present disclosure relates generally to sequencing techniques, and more specifically to methods, systems, devices, and non-transitory computer-readable storage media for processing images of biological samples (e.g., to obtain sequencing data). An exemplary method of determining nucleic acid sequences of a plurality of sequencing colonies comprises: obtaining an input image of a surface, wherein the plurality of sequencing colonies are attached to the surface; detecting a set of sequencing colonies of the plurality of sequencing colonies in the input image; executing in parallel, using a graphics processor, a plurality of iterative processes to obtain signal amplitudes for the detected set of sequencing colonies, wherein each iterative process corresponds to a respective detected sequencing colony in the set; and determining, at least partially based on the signal amplitudes for the detected set of sequencing colonies, portions of nucleic acid sequences of the plurality of sequencing colonies.

The present disclosure relates generally to sequencing techniques, and more specifically to methods, systems, devices, and non-transitory computer-readable storage media for processing images of biological samples (e.g., to obtain sequencing data). An exemplary method of determining nucleic acid sequences of a plurality of sequencing colonies comprises: obtaining an input image of a surface, wherein the plurality of sequencing colonies are attached to the surface; detecting a set of sequencing colonies of the plurality of sequencing colonies in the input image; executing in parallel, using a graphics processor, a plurality of iterative processes to obtain signal amplitudes for the detected set of sequencing colonies, wherein each iterative process corresponds to a respective detected sequencing colony in the set; and determining, at least partially based on the signal amplitudes for the detected set of sequencing colonies, portions of nucleic acid sequences of the plurality of sequencing colonies.

This disclosure provides methods and devices for the label-free detection of target molecules of interest. The principles of the disclosure are particularly applicable to the detection of biological molecules (e.g., DNA, RNA, and protein) using tandard SiO.sub.2-based microarray technology.