A method of counting individual optically active dye labels immobilized on a substrate, the method comprising: at an electronic device with one or more processors and memory: (a) obtaining digital image data corresponding to a two-dimensional digital image of the substrate, wherein the digital image data comprise a two-dimensional array of intensity values, where each intensity value corresponds to a particular location on the substrate; (b) identifying a first set of regions of the digital image data by segmenting the digital image data into multiple regions based on the intensity values, wherein each region comprises an average, median, or maximum intensity value which is above a predefined threshold; (c) for each region of the digital image data: increasing a count of optically active dye labels by one; and identifying a peak location of intensity values from an optically active dye label, and subtracting a reference signal distribution from the intensity values at the identified peak location to obtain adjusted intensity values for the region, wherein the reference signal distribution corresponds to a point spread function of a single optically active dye label; (d) identifying a second set of one or more sub-regions of the adjusted intensity values for each of said regions by segmenting the adjusted intensity values for each of said regions into one or more sub-regions based on the adjusted intensity values, wherein, if the average, median, or maximum adjusted intensity value in any of said sub-regions is above the predefined threshold, (e) for each of said sub-regions: increasing the count of the optically active dye labels by one. (a) obtaining digital image data corresponding to a two-dimensional digital image of the substrate, wherein the digital image data comprise a two-dimensional array of intensity values, where each intensity value corresponds to a particular location on the substrate; (b) identifying a first set of regions of the digital image data by segmenting the digital image data into multiple regions based on the intensity values, wherein each region comprises an average, median, or maximum intensity value which is above a predefined threshold; (c) for each region of the digital image data: increasing a count of optically active dye labels by one; and identifying a peak location of intensity values from an optically active dye label, and subtracting a reference signal distribution from the intensity values at the identified peak location to obtain adjusted intensity values for the region, wherein the reference signal distribution corresponds to a point spread function of a single optically active dye label; increasing a count of optically active dye labels by one; and identifying a peak location of intensity values from an optically active dye label, and subtracting a reference signal distribution from the intensity values at the identified peak location to obtain adjusted intensity values for the region, wherein the reference signal distribution corresponds to a point spread function of a single optically active dye label; (d) identifying a second set of one or more sub-regions of the adjusted intensity values for each of said regions by segmenting the adjusted intensity values for each of said regions into one or more sub-regions based on the adjusted intensity values, wherein, if the average, median, or maximum adjusted intensity value in any of said sub-regions is above the predefined threshold, (e) for each of said sub-regions: increasing the count of the optically active dye labels by one.