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Second Harmonic Detection of Sinusoidally Modulated Two-Photon Excited Fluorescence

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Abstract

Three interrelated problems that are routinely encountered in the taking of two-photon excitation spectra are inherently low signal levels, interferences arising from one-photon interactions such as scatter (Mie, Rayleigh, Raman) and absorption, and the need to correct the source for intensity and pulse width fluctuations. As a means of separating the quadratic signal of interest from linear interferences our group has traditionally used a linear regression method (1). This approach, though effective, suffers in two areas. The primary limitation is that it requires at least five measurements in order to yield meaningful results. This entails the attenuation of an already weak signal. A secondary concern is that the regression is performed at each wavelength resulting in excessively long data collection time; typically 6 to 8 hours for a single spectrum.

© 1990 Optical Society of America

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