Abstract
Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.
Highlights
We built a semi-automated device from affordable commercial components, which is able to complete a delicate task currently carried out by skillful experts trained to do difficult manipulations on a microscope
To isolate single cells from suspension we used a motorized microscope and micropipette controlled by computer vision
Combination of 1 μ m positioning precision, adaptive cell targeting, and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation
Summary
We built a semi-automated device from affordable commercial components, which is able to complete a delicate task currently carried out by skillful experts trained to do difficult manipulations on a microscope. Manual single cell isolation for DNA/RNA sequencing from a suspension with a mouth micropipette is a precise but very low throughput method requiring a well-trained expert[4]. Cell encapsulation in nano- or picoliter-scale droplets[18,21,22] is a promising route for single cell manipulations in water-oil-based two-phase microfluidic systems. It could not gain extensive use, probably due to the technical challenges of operating complex microfluidic chips. A robot with computer vision-based feedback and closed-loop process control was demonstrated to sort single cells[19] This system used initially immobilized cells, and bright-field illumination was critically needed for the closed-loop motion control of the micropipette. The current study is limited to the isolation of mammalian cells, insect, plant and yeast cells can be isolated by a micropipette as it is carried out manually in many laboratories
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