Methods of penning micro-objects using positive dielectrophoresis

1 . 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.

2 . The method of claim 1 , wherein the projected light of the first light beam has a substantially circular, oblong, triangular, rectangular, square, polygonal, or irregular shape, and completely illuminates the first micro-object.

3 . The method of claim 1 , wherein the projected light of the second light beam forms a line having a substantially circular, oblong, triangular, rectangular, square, polygonal, or irregular shape.

4 . The method of claim 3 , wherein the width of the line formed by the second light beam is between about 5 microns and about 15 microns.

5 . The method of claim 1 , wherein the projected light of the second light beam has a shape that encloses the first light beam, wherein there is a space between the first and second light beams, and wherein any illumination of the space between the first and second light beams is insufficient to activate DEP electrodes located in the space between the first and second light beams.

6 . The method of claim 1 , wherein moving the first micro-object from the first position to the third position on the electrode activation substrate isolates the first micro-object from other micro-objects located in the microfluidic device.

7 . The method of claim 1 , wherein the light projected upon the first position by the first light beam has a length, width, or diameter that is smaller than a width of the opening of the sequestration pen.

8 . The method of claim 1 , wherein the light projected upon the second position by the second light beam defines a shape having a length, width, or radius that is about 40 microns to about 80 microns, and wherein the length, width, or radius of the defined shape increases during a period of time defined by an initial time when the first and second light beams are first projected upon the electrode activation substrate, and a subsequent time when the projected light of the first light beam arrives at the third position on the electrode activation substrate or arrives at the opening of the sequestration pen of the microfluidic device.

9 . The method of claim 1 , wherein the projected light of the second light beam changes shape once a portion of the projected light of the second light beam reaches or enters the opening of the sequestration pen of the microfluidic device.

10 . The method of claim 9 , wherein the shape of the projected light of the second light beam continues to change as the projected light of the first light beam approaches and/or enters the opening of the sequestration pen.

11 . The method of claim 1 , further comprising projecting light from a third light beam upon a fourth position on the electrode activation substrate, wherein the fourth position is located in or proximal to the opening of the sequestration pen fluidically connected to the flow region or a microfluidic channel.

12 . The method of claim 1 , wherein the first micro-object is a bacterial cell, a fungal cell, a yeast cell, an animal cell, and/or a mammalian cell, or wherein the first micro-object is a capture bead.

13 . A system for performing a method of selectively positioning a first micro-object in a microfluidic device, the system comprising:

an optical train having a structured light source; and

a master controller having a control module with a processor and a non-transitory computer accessible storage medium storing thereupon a sequence of instructions which, when executed by the processor of the control module, causes the control module to perform the method of claim 1 .

14 . The system of claim 13 , further comprising a media module, a motive module, an imaging module, and/or a tilting module.

15 . The method of claim 7 , wherein the light pattern projected by the second light beam has at least one dimension larger than the opening of the sequestration pen.

16 . The method of claim 1 , wherein the first light beam moves the captured first micro-object into the sequestration pen.