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Single cell deposition and patterning with a robotic system.

Lu Z, Moraes C, Ye G, Simmons CA, Sun Y - PLoS ONE (2010)

Bottom Line: By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location.A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells.Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

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Augmented parallel deposition.(A) Schematic outline outlining the process for populating an array of microwells with single cells. A dense cell suspension is pipetted onto the microwell array, and allowed to settle. Excess cells are then washed away. (B) Comparison of single cell trapping efficiencies between two washing methods for three well diameters in the microwell array. (C) Fluorescently labeled single cells trapped in microwell arrays via random deposition. (D) Robotic manipulation of single cells was used to correct errors in the randomly seeded array.
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pone-0013542-g005: Augmented parallel deposition.(A) Schematic outline outlining the process for populating an array of microwells with single cells. A dense cell suspension is pipetted onto the microwell array, and allowed to settle. Excess cells are then washed away. (B) Comparison of single cell trapping efficiencies between two washing methods for three well diameters in the microwell array. (C) Fluorescently labeled single cells trapped in microwell arrays via random deposition. (D) Robotic manipulation of single cells was used to correct errors in the randomly seeded array.

Mentions: Microwells can be used as a patterning tool to transfer single cells to other substrates [13]. During this process, a dense suspension of cells is deposited on a surface patterned with microwells, and allowed to settle into the microwells. Excess cells are then washed away (Figure 5A), and the chip can be flipped over to transfer the patterned cells onto any desired substrate. This technique, dubbed the “BioFlipChip” [13], is capable of positioning a large number of single cells simultaneously. As has been reported previously [14] and observed in the present study, the single cell trapping efficiency with microwell arrays is dependent on well height and diameter. However, we found that the efficiency is also strongly dependent on washing method (Figure 5B), and qualitatively, upon operator skill and proficiency. A skilled operator was unable to obtain more than an 80% trapping efficiency with either washing method, confirming previous findings [14]. A sample image of an array section is shown in Figure 5C, in which ∼80% of the wells trapped single cells. The robotic manipulation system was used after this random deposition of cells to fill in empty sites and remove extra cells, increasing single cell trapping efficiency to 100% (Figure 5D). This “post-processing” technique maintains the established advantages in speed obtained in using the BioFlipChip approach, while improving the overall efficiency of patterning single cells.


Single cell deposition and patterning with a robotic system.

Lu Z, Moraes C, Ye G, Simmons CA, Sun Y - PLoS ONE (2010)

Augmented parallel deposition.(A) Schematic outline outlining the process for populating an array of microwells with single cells. A dense cell suspension is pipetted onto the microwell array, and allowed to settle. Excess cells are then washed away. (B) Comparison of single cell trapping efficiencies between two washing methods for three well diameters in the microwell array. (C) Fluorescently labeled single cells trapped in microwell arrays via random deposition. (D) Robotic manipulation of single cells was used to correct errors in the randomly seeded array.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958835&req=5

pone-0013542-g005: Augmented parallel deposition.(A) Schematic outline outlining the process for populating an array of microwells with single cells. A dense cell suspension is pipetted onto the microwell array, and allowed to settle. Excess cells are then washed away. (B) Comparison of single cell trapping efficiencies between two washing methods for three well diameters in the microwell array. (C) Fluorescently labeled single cells trapped in microwell arrays via random deposition. (D) Robotic manipulation of single cells was used to correct errors in the randomly seeded array.
Mentions: Microwells can be used as a patterning tool to transfer single cells to other substrates [13]. During this process, a dense suspension of cells is deposited on a surface patterned with microwells, and allowed to settle into the microwells. Excess cells are then washed away (Figure 5A), and the chip can be flipped over to transfer the patterned cells onto any desired substrate. This technique, dubbed the “BioFlipChip” [13], is capable of positioning a large number of single cells simultaneously. As has been reported previously [14] and observed in the present study, the single cell trapping efficiency with microwell arrays is dependent on well height and diameter. However, we found that the efficiency is also strongly dependent on washing method (Figure 5B), and qualitatively, upon operator skill and proficiency. A skilled operator was unable to obtain more than an 80% trapping efficiency with either washing method, confirming previous findings [14]. A sample image of an array section is shown in Figure 5C, in which ∼80% of the wells trapped single cells. The robotic manipulation system was used after this random deposition of cells to fill in empty sites and remove extra cells, increasing single cell trapping efficiency to 100% (Figure 5D). This “post-processing” technique maintains the established advantages in speed obtained in using the BioFlipChip approach, while improving the overall efficiency of patterning single cells.

Bottom Line: By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location.A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells.Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Integrating single-cell manipulation techniques in traditional and emerging biological culture systems is challenging. Microfabricated devices for single cell studies in particular often require cells to be spatially positioned at specific culture sites on the device surface. This paper presents a robotic micromanipulation system for pick-and-place positioning of single cells. By integrating computer vision and motion control algorithms, the system visually tracks a cell in real time and controls multiple positioning devices simultaneously to accurately pick up a single cell, transfer it to a desired substrate, and deposit it at a specified location. A traditional glass micropipette is used, and whole- and partial-cell aspiration techniques are investigated to manipulate single cells. Partially aspirating cells resulted in an operation speed of 15 seconds per cell and a 95% success rate. In contrast, the whole-cell aspiration method required 30 seconds per cell and achieved a success rate of 80%. The broad applicability of this robotic manipulation technique is demonstrated using multiple cell types on traditional substrates and on open-top microfabricated devices, without requiring modifications to device designs. Furthermore, we used this serial deposition process in conjunction with an established parallel cell manipulation technique to improve the efficiency of single cell capture from ∼80% to 100%. Using a robotic micromanipulation system to position single cells on a substrate is demonstrated as an effective stand-alone or bolstering technology for single-cell studies, eliminating some of the drawbacks associated with standard single-cell handling and manipulation techniques.

Show MeSH
Related in: MedlinePlus