TIME-RESOLVED, TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY OF CULTURED-CELLS USING A TB CHELATE LABEL

A total internal reflection fluorescence microscope with time-resolved spectroscopy and CCD imaging capabilities is described. The capabilit y of the microscope to selectively detect, via temporal resolution, th e presence of a long-lived luminescence label in biological cells in v itro was evaluated. To chelates having radiative lifetimes of ca. 1.5 ms were employed as long-lived, extrinsic labels. Microspectroscopy an d imaging were performed on mouse 3T3 cells stained with To chelates, stained with DiI (an extrinsic membrane label of ns lifetime), and in the absence of an extrinsic label. The results demonstrate the efficie ncy of using time-resolved detection to discriminate against intrinsic and extrinsic sources of short-lived emission in cultured cells, whic h enables long-lived, extrinsic luminescence to be selectively detecte d. Potential applications of this technology are discussed.