A microfluidic device comprising an enclosure comprising: a base, a microfluidic circuit structure disposed on the base, and a cover which collectively define a microfluidic circuit, wherein said microfluidic circuit comprises: a flow region configured to contain a flow of a first fluidic medium; one or more inlets through which said first medium can be input into said flow region; one or more outlets through which said first medium can be removed from said flow region; a microfluidic channel comprising at least a portion of said flow region; and a microfluidic sequestration pen (436, 438, 440) comprising: an isolation region for containing a second fluidic medium, the isolation region having a single opening; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel having a width W con between about about 20 microns to about 100 microns and a distal opening into said isolation region, and wherein a length L con of said connection region from said proximal opening to said distal opening is as least 1.0 times a width W con of said proximal opening of said connection region, wherein said width W con of said connection region at the proximal opening is parallel to a direction of said flow of said first medium through said flow region, such that said isolation region of said microfluidic sequestration pen is an unswept region of said micro-fluidic device. a flow region configured to contain a flow of a first fluidic medium; one or more inlets through which said first medium can be input into said flow region; one or more outlets through which said first medium can be removed from said flow region; a microfluidic channel comprising at least a portion of said flow region; and a microfluidic sequestration pen (436, 438, 440) comprising: an isolation region for containing a second fluidic medium, the isolation region having a single opening; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel having a width W con between about about 20 microns to about 100 microns and a distal opening into said isolation region, and wherein a length L con of said connection region from said proximal opening to said distal opening is as least 1.0 times a width W con of said proximal opening of said connection region, an isolation region for containing a second fluidic medium, the isolation region having a single opening; and a connection region fluidically connecting said isolation region to said flow region, wherein said connection region comprises a proximal opening into said microfluidic channel having a width W con between about about 20 microns to about 100 microns and a distal opening into said isolation region, and wherein a length L con of said connection region from said proximal opening to said distal opening is as least 1.0 times a width W con of said proximal opening of said connection region, wherein said width W con of said connection region at the proximal opening is parallel to a direction of said flow of said first medium through said flow region, such that said isolation region of said microfluidic sequestration pen is an unswept region of said micro-fluidic device.