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ANALYTICAL CHEMISTRY, VOL. 62, NO. 9, MAY 1, 1990
product of the filter transmission and photocathode efficiency is calculated by
(7) where F(X) is the fluorescence emission spectrum, T(X) is the measured transmission spectrum of the filter combination, and Q(X) is the quantum yield spectrum of the photocathode as specified by the manufacturer. These spectra were digitized and the integrals were calculated numerically to give r f ( h ) = 0.071. Given the average power of 245 mW and a peak power estimated to be 600 W for a 5-ps pulse width, the expected count rate per nM is estimated to be 5600. The actual count rate per nM is 74, which is a factor of 76 smaller. The most likely factors responsible for the missing signal in the experiment are (1)the nonzero size of the image and (2) spherical aberration in both lenses. It is possible that even higher sensitivity can be achieved by further improving the optical scheme. Two-photon spectroscopy is intrinsically suited to small volume measurements because minimization of the path length provides a small probe volume. If a diffraction-limited lens were used to focus the beam to a 5-pm spot, the excitation efficiency would have been greater than that obtained in this work. For the 120 pM detection limit reported here, only 5000 molecules would be present in the 50-pL diffraction-limited probe volume at any one time, accounting for the additional 27% of the fluorescing molecules outside of 42,. For the study of small numbers of molecules, this volume is larger and the concentration detection limit is several orders of magnitude worse than that obtained in one-photon excited fluorescence
spectroscopy for rhodamine 6G ( 1 3 , 1 4 ) . However, the laser source used for the two-photon excitation is modest by the standards of nonlinear spectroscopy: the excitation efficiency would be increased 50-fold by use of a commercially available synchronously pumped, cavity-dumped dye laser. The achievable two-photon excitation efficiency will continue to increase with technological advances, such as higher power ultrafast lasers. Two-photon fluorometry is valuable for probing microenvironments of interesting media. The high detectability reported here makes two-photon excited fluorometry applicable to studies of dilute probes in surfaces, thin films, micelles, and membranes.
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RECEIVED for review November 2, 1989. Accepted January 19, 1990. This work was supported by the National Science Foundation under Grant CHE-8814602.
CORRECTION Determination of Serum Cholesterol by a Modification of the Isotope Dilution Mass Spectrometric Definitive Method Polly Ellerbe, Stanley Meiselman, Lorna T. Sniegoski, Michael J. Welch, and Edward White V (Anal. Chem. 1989, 61, 1718-1723). Reference 6 should read as follows: Ellerbe, P.; Meiselman, S.; Cohen, A.; Schaffer, R.; Welch, M. J.; White, E., V. Presented at the 34th Annual Conference on Mass Spectrometry and Allied Topics, Cincinnati, OH, June 8-13. 1986.
