1683
Anal. Chem. 1985, 57, 1663-1669
Reduction of Raman Background in Laser-Induced Fluorescence by Second Harmonic Detection Mitchell Trkula and Richard A. Keller* Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
Harmonic generation in saturated fluorescence Is shown to reduce the scattered light background in laser-induced fluorescence. I n the harmonic generation technlque, the fluorescence is excited with sinusoidal amplitude modulation and the nonlinear response of saturated fluorescence generates harmonics of the excitation frequency. Detection at the second harmonic is shown to completely suppress the amplitude of Raman scatterlng background. The suppression occurs with no decrease in background noise for detection systems at the statlstlcal noise ilmit and, since second harmonic detection decreases the fluorescence signal, signalto-noise is degraded. For systems with nonstatisticai noise, the noise in the background is decreased with second harmonic detectlon, and improved analytical detectlon limits may result. Second harmonic detectlon in fluorescence spectrometry is shown to be capable of suppressing interferlng scattering features which are up to 150 times more Intense than the underlying fluorescence. Fluorescence spectra from a steady-state concentration of 210 molecules in the probe volume are shown. A major benefit in optical alignment of the Instrument is obtained through second harmonic detectlon.
Frueholz and Gelbwachs, namely, they qualitatively showed that detection at the sum or difference frequency enhanced the signal-to-background ratio and improved the S I N . A different type of nonlinear response in an absorption analysis has been applied to liquid chromatographic detection by Pang and Morris (7).In their experiments, a thermal lens gives rise to a signal at the second harmonic of the laser modulation frequency. Their results show a slight degradation of S I N when harmonic detection is compared to two-laser, pump-probe methods; however, the use of a single laser avoids the critical alignment problem of the pump-probe technique. Our interest in these techniques stems from work toward achieving true single molecule detection. In our experimental apparatus, the detection-limiting noise arises from the Raman scatter of the solvent which is present in the probe volume (3). Hence, it appeared that intermodulated fluorescence or harmonic generation might be used to advantage. Also, since previous studies had dealt only with atomic fluorescence in the gas phase, it was of interest to see how successful these techniques would be with a molecular system in liquid solution which requires a much higher irradiance to saturate.
THEORETICAL BACKGROUND The fluorescence response of a two-level system when excited by a laser of irradiance I ( t ) is given by (8)
Laser-induced fluorescence is a powerful and sensitive technique in both chemical and biological analysis. For a recent review of this subject, see ref 1. The technique has been used to detect single atoms in the gas phase (2) and is predicted to be capable of detecting single molecules in the fluid phase (3). It is often the case for this extremely sensitive technique that the limiting noise in the background arises from scattering processes which occur in the same spectral region as the fluorescence signal. An important difference between fluorescence and scattering (Raleigh, Raman, Mie) is that fluorescence response is a nonlinear function of the irradiance while scattering is linear. In this paper we demonstrate that the nonlinearity in the fluorescence response can be used to advantage in ultrasensitive analysis. The nonlinear response of saturated fluorescence is a well-known and well-studied phenomenon. In 1972 Sorem and Schawlow used this nonlinearity to develop intermodulated fluorescence to study sub-Doppler spectra of atomic and molecular systems (4). However, it was not until recently, with the work of Frueholz and Gelbwachs ( 5 ) ,that this property was utilized from an analytical point of view. In their experiments, a sinusoidally modulated laser was used to excite sodium fluorescence in an air-acetylene flame. In this case the nonlinear response of the fluorescence generated harmonics of the excitation frequency. They showed clearly that detection of the fluorescence signal at the second harmonic of the laser modulation frequency results in a greatly reduced background and a factor of 5.3 increase in signal-to-noiseratio ( S I N ) . In a similar experiment, Omenetto, Hart, and Wineforder (6) used counterpropagating beams modulated at two different frequencies to excite sodium fluorescence in a Ar-02-H2 flame, Le., the classical intermodulated fluorescence experiment. Their results were similar to those of
where Io is the saturation irradiance and 0 is a proportionality constant. Io can be written in terms of the rate constants for excitation and deexcitation (8). Experimentally, Io is determined from a plot of the inverse of eq 1
-1- - - Io F(t) W(t)
1 +
ii
which is a straight line whose slope-to-intercept ratio gives 1,. At low irradiance, when II(t)l
