Figure 2. Flow chart of the automated single-cell analysis and isolation system.

Approximately 5.0 × 10⁴ cells in culture medium were added to the microchamber array chip equipped with an aluminum frame (step (a)) and then introduced into 30-μm PDMS microchambers by brief centrifugation (50 × g, at room temperature for 1 min) (step (b)). The microchamber array was scanned with a CCD camera on the robot. Based on the fluorescent image, microchambers containing no or more than 2 fluorescent particles were excluded from further analyses. Green circles, excluded microchambers; red circles, selected microchambers (step (c)). A histogram of fluorescent intensity from each cell was generated for permutation of cells by the order of fluorescent intensities, a list of addresses, fluorescent intensities and images, as well as a transmission image of each cell was generated (step (d)). Cells of interest were automatically retrieved with a glass capillary attached to the micromanipulator. Recovery of each cell was repeated until the fluorescence from each microchamber was absent (step (e)). Each retrieved cell was transferred to an assigned reservoir well (step (f)). Scale bars = 200 μm (b) and 30 μm (e).