Passive optical separation within a 'nondiffracting' light beam.

ABSTRACT

A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam-the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.