CELLULAR SPECTROSCOPY AND MULTIANGLE LIGHT SCATTERING BY FLOW CYTOMETRY: OPTICAL TEST BENCH AS A DEVELOPMENTAL TOOL

摘要

We designed and fabricated a flow cytometry work bench for the purpose of testing optical schemes utilized in two specialized flow cytometry techniques. Fluorescent spectroscopy and multi-angle scattering of laser light has potential for generating novel classes of cell population information. The acquisition of full fluorescence spectrum of dye bound to living cells may associate specific function to spectral shift. Similarly, detection of 2 discrete angles of light scatter was shown to describe internal structures of cells based on refractive and diffractive properties. We utilized a flow cytometry electro- cells based on refractive and diffractive properties. We utilized a flow cytometry electro- optical test bench to develop and describe alternate light collection schemes for these two techniques. Mouse bone marrow was chosen as the subject of analysis because it contains a consistent mix of cells that possess distinct biophysical properties. More importantly, these cells are the source of various biological systems that are targeted by environmental stress and clinical disease. Hoechst 33342 fluorescent dye binds strongly to nucleic pairs within the cell nucleus but is also retained within the outer cytoplasm were it functions as a substrate for a class of ATP driven efflux proteins associated with multi-drug resistance. Measurements of the efflux of this probe from the cell interior have been used extensively to identify and separate potential stem cells in human and mouse systems We acquired full spectrum signatures of bound and non-bound Hoechst by mounting a scanning monochrometer to a flow cytometer. Resulting spectra from bound Hoechst 33342 indicated 2 alternate emission peaks besides the primary unbound dye emission curve suggesting differential binding of potentially important precursor cells. Flow cytometry light scatter produces a well known pattern of RBC, lymphoid and granular populations from mouse bone marrow cells. We built a 2 element scatter detector to measure discrete angles of scattered light and made a correlation between refractive and diffractive sub-cellular properties.