Optical selection and patterning of cells
The ability to select a small number of cells from a heterogeneous population is fundamental to many aspects of biological research. Selections form the basis of genetic screens, of protein engineering and directed evolution, and of protocols to produce stably transformed or genome-edited cell lines. In many instances, one would like to select cells on the basis of complex dynamic or morphological features. For example, in a culture of olfactory neurons, one might screen for calcium flux in response to a specific odorant; and then wish to select responsive cells for subsequent transcriptional profiling. Or in a culture with single genes knocked down by an siRNA library, one might find cells with unusual shapes, organelle sizes, or metabolic responses; and then wish to select these cells to determine which gene had been knocked down. These types of selections are difficult to perform with existing tools.
Photostick |
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Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. Figure: Components of the photostick protocol. (A) Sequence of steps in photostick method. Photochemical immobilization retains target cells while others are washed away under mild protease treatment. (B) Trifunctional crosslinkers Cy3- and Cy5-SBED for photochemical immobilization with simultaneous fluorescent labeling and biotynilation. (C) Digital micromirror device (DMD) for patterned violet illumination to activate photo-crosslinker. See: M.P. Chien, C.A. Werley, S.L. Farhi, A.E. Cohen, Photostick: a method for selective isolation of target cells from culture, Chemical Science, 6, 1701-1705, 2015. |