Methods of penning micro-objects using positive dielectrophoresis

A method of selectively positioning a first micro-object in a microfluidic device comprising an enclosure having an inlet, an outlet, a flow region connecting the inlet and the outlet, and an electrode activation substrate having a photoconductive layer, wherein the enclosure of the microfluidic device further comprises a sequestration pen, and wherein the sequestration pen opens laterally from the flow region, the method comprising:

• projecting light from a first light beam upon a first position on the electrode activation substrate, wherein the first position is proximal to the first micro-object, and wherein the projected light of the first light beam activates a positive dielectrophoresis (DEP) force within the enclosure sufficient to capture the first micro-object;
• projecting light from a second light beam upon a second position on the electrode activation substrate, wherein the second position is adjacent to or at least partially surrounds the first position, without overlapping the first position, the projected light of the second light beam activating a positive DEP force within the enclosure sufficient to capture at least one second micro-object other than the first micro-object, and wherein the projected light of the second light beam has a non-uniform width;
• moving the projected light of the first light beam towards a third position on the electrode activation substrate, wherein the third position on the electrode activation substrate is located within the sequestration pen, wherein the DEP force activated by the projected light of the first light beam is sufficient to move the first micro-object to the-third position;
• and
• moving the projected light of the second light beam in relation to the projected light of the first light beam to move the at least one second micro-object away from the third position, wherein the thinner region of the second light beam is oriented towards an opening of the sequestration pen as the projected light of the second light beam moves in relation to the projected light of the first light beam.