PubMed · 15603422
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Jurek W Dobrucki. 2004. Confocal microscopy: quantitative analytical capabilities.. https://doi.org/10.1016/s0091-679x(04)75003-7
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We present a resonant optical nanoantenna positioned at the end of a metal-coated glass fiber near-field probe. Antenna resonances, excitation conditions, and field localization are directly probed in the near field by single fluorescent molecules and compared to finite integration technique simulations. It is shown that the antenna is equivalent to its radio frequency analogue, the monopole antenna. For the right antenna length and local excitation conditions, antenna resonances occur that lead to an enhanced localized field near the antenna apex. Direct mapping of this field with single fluorescent molecules reveals a spatial localization of 25 nm, demonstrating the importance of such antennas for nanometer resolution optical microscopy.
Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a better understanding of the signaling pathways that it modulates. The method presented here utilizes 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and a FLEXstation for the continuous measure of PP2A activity associated with many different protein preparations. This automated fluorescence-based assay offers several distinct advantages over colorimetric and radioactive assays of phosphatase activity including (1) decreased substrate preparation time, (2) real-time kinetic data, (3) high sensitivity, and (4) the capability to analyze a wide variety of phosphatases.