Single Cell Optical Manipulations

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Characterization of biological samples relies heavily on microscopy where, in response to various stimuli, molecular probes and an impressive list of contrast reagents can be used to identify and label live cells of interest. These methods, such as transfection of genes encoding fluorescent proteins, the use of membrane-permeable dyes or antibody labeling require prior knowledge of cellular markers or use of elaborate reporter constructs. However, based solely on visual inspection or using image processing algorithms, it is possible to distinguish cells which exhibit distinct biological properties from among thousands of counterparts within a microscopy field. Indeed, a large number of critical biological processes are determined by cellular characteristics that can only be assessed upon direct microscopy observation. Such visual phenotypes often have major and well-understood molecular and biological implications but lack practical biochemical markers for experimental research. For example, cell movement is essential in normal embryonic development, wound healing and immune responses. Cell shape is associated with growth, division and death, as cytoskeletal properties determine cell differentiation, essential in tissue morphogenesis and disease. Furthermore, the position of cells regarding distance to chemical cues and cell-to-cell contact are critical in chemokinesis, neural function and immune responses. The overarching goal of my NSERC Discovery program is focused on technology developments that allow identifying, tagging, manipulating, capturing and expanding cells based on visual information that can only be obtained by microscopy imaging. The tools I propose to achieve this are based on recent technological developments from my lab, such as Cell Labeling via Photobleaching (CLaP) which can tag individual cells upon observation. CLaP uses laser irradiation for crosslinking biotin on the plasma membrane of living cells and fluorescent streptavidin conjugates to interact with them in different ways. For the next five years I propose three objectives that comprise the exploitation of CLaP, profiting from different capacities of the technology and extending its applications. 1) To develop single cell CLaP-based gene delivery. CLaP can be combined with virus-based technology to select with a laser the individual cells in a microscopy field to be transfected. 2) To generate and test CLaP oligonucleotide barcodes for single cell sequencing. CLaP can be used to bind predefined biotinylated DNA sequences to individual cells in culture. These DNA tags are bound to the plasma membrane using laser illumination and only to selected individual cells. Labeled cells will be identified after sequencing. 3) To use CLaP to derive novel cell populations from visual phenotypes. I will use cell motility as the basis to create novel cell lines that will allow uncovering of molecular mechanism normally hidden in large cell ensembles.