Response function, light scattering instrument

Heyder, J., and J. Gebhart, 1979. Optimization of response functions of light scattering instruments for size evaluation of aerosol particles, Appl. Opt., 18, 705-711. 

Our experiments are typically carried out at DNA concentrations of 20-50 /ig/ml with 1 ethidium per 300 bp, so that depolarization by excitation transfer is negligible.(18) The sample is excited with 575-nm light, and the fluorescence is detected at 630, 640, or 645 nm. Less than one fluorescent photon is detected for every 100 laser shots. The instrument response function e(t) is determined using 575-nm incident light scattered from a suspension of polystyrene latex spheres. 

Instrumentation. The steady-state fluorescence spectra were measured with Perkin-Elmer MPF-44B fluorescence spectrophotometer. The single-photon counting instrument for fluorescence lifetime measurements was assembled in-house from components obtained from EG G ORTEC. A PRA-510B light pulser filled with gas was used as the excitation source. Instrument response function was obtained with DuPont Ludox scatter solution at the excitation wavelength. 

Two instruments considered by Cooke and Kerker (1975) had single-valued response functions, presumably because of broad-band (white) light sources and large angular apertures for both incident and scattered light. 

Examples of the instrument response function and observed fluorescence are shown in Figure 3. The data set in the top plot of F%ure 3 was obtained by observing light scattered from a glucose solution with both the output monochromator of the dye laser and the emission monochromator set to 366 nm. Thus this represents the instrument s intrinsic response. Note that the full width at half the maximum height of the peak (FWHM) is about 2 ns, greater than the laser s quoted 200-300 ps this implies that the detection electronics do not respond instantaneously on this time scale. The lower plot of Figure 3 shows the observed fluorescence from 10 pM dansylamide, with excitation at 366 nm and emission monitored at 560 nm. The monoexponential lifetime was determined to be about 2.5 ns. 

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