An automated method of detecting an assay-positive assay area for an assay of an analyte of interest with a reagent within a microfluidic device (100; 240) comprising circuit elements comprising: a microfluidic channel (122; 522); and a sequestration pen (124, 126, 128, 130; 244, 246, 248; 526, 528) comprising an isolation region having a single opening, and a connection region (254; 560, 562, 564), said connection region having a proximal opening to the microfluidic channel and a distal opening to the isolation region, and wherein the isolation region of the sequestration pen is an unswept region of the microfluidic device that experiences substantially no flux of fluid when fluid is flowing through the microfluidic channel, the method comprising: automatically identifying an assay area AA x (570, 572; 574, 576; 578, 579) based, at least in part, on the dimensions of the circuit elements; collecting a set of digital images I i, i=1 to n, of the automatically-identified assay area AA x; calculating a rate of change Δ x of at least one parameter characterizing a distribution of light intensity values for a set of pixels that make up each image I i of the automatically-identified assay area AA x over the course of all or part of the assay based on the set of digital images I i of the automatically-identified assay area AA x; comparing the rate of change Δ x to a threshold value Δ o; and determining that the automatically-identified assay region AA x is assay-positive if Δ x is greater than Δ o. a microfluidic channel (122; 522); and a sequestration pen (124, 126, 128, 130; 244, 246, 248; 526, 528) comprising an isolation region having a single opening, and a connection region (254; 560, 562, 564), said connection region having a proximal opening to the microfluidic channel and a distal opening to the isolation region, and wherein the isolation region of the sequestration pen is an unswept region of the microfluidic device that experiences substantially no flux of fluid when fluid is flowing through the microfluidic channel, the method comprising: automatically identifying an assay area AA x (570, 572; 574, 576; 578, 579) based, at least in part, on the dimensions of the circuit elements; collecting a set of digital images I i, i=1 to n, of the automatically-identified assay area AA x; calculating a rate of change Δ x of at least one parameter characterizing a distribution of light intensity values for a set of pixels that make up each image I i of the automatically-identified assay area AA x over the course of all or part of the assay based on the set of digital images I i of the automatically-identified assay area AA x; comparing the rate of change Δ x to a threshold value Δ o; and determining that the automatically-identified assay region AA x is assay-positive if Δ x is greater than Δ o. automatically identifying an assay area AA x (570, 572; 574, 576; 578, 579) based, at least in part, on the dimensions of the circuit elements; collecting a set of digital images I i, i=1 to n, of the automatically-identified assay area AA x; calculating a rate of change Δ x of at least one parameter characterizing a distribution of light intensity values for a set of pixels that make up each image I i of the automatically-identified assay area AA x over the course of all or part of the assay based on the set of digital images I i of the automatically-identified assay area AA x; comparing the rate of change Δ x to a threshold value Δ o; and determining that the automatically-identified assay region AA x is assay-positive if Δ x is greater than Δ o.