PubMed · 5078964
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R P Parker. 1972. Semiconductor detectors. Introduction.. https://pubmed.ncbi.nlm.nih.gov/5078964/
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beta-Emitting 90Sr is used with a plastic scintillator to produce excitation-light pulses for fluorescence lifetime analysis. This light source is less expensive, more compact, and much more reliable than traditionally employed excitation sources such as lasers or pulsed flash lamps. The pulse train from this light source varies randomly in amplitude and time. Cross-correlation signal analysis is ideal for such a source because, unlike other time domain techniques, cross correlation takes complete advantage of its random nature. Here we report on the construction of an instrument and the methods employed to make fluorescence lifetime measurements via the new source and an analog correlation processor. Although the light intensity of the scintillator-based excitation source is comparatively low, an adequate signal level can be generated. The fluorescence lifetimes of three fluorophores are measured with a 1-mCi radionuclide to demonstrate a lifetime range from less than 1.5 to 28 ns. Long-lifetime measurements require an extra calibration step in order to compensate for delay cable energy loss. The light collection efficiency of the current instrument was found to be undesirably low; improvements in the instrument optics are suggested that will increase the collection efficiency and enhance the detection capability.
We report the first observation of two-photon excitation of fluorescence using the evanescent wave from total internal reflectance (TIR). The evanescent wave at 770 nm from a fs Ti:Sapphire laser was used to excite the calcium probe Indo-1 at a quartz-water interface. The emission intensity of Indo-1 depended quadratically on the incident power at 770 nm, when incidence angles were above and below the critical angle (theta c) for TIR. The time-resolved intensity and anisotropy decays with TIR at 770 nm demonstrated the origin of the signals as Indo-1 and eliminated the possibility of scattered light contributing to the signal. The emission from Indo-1 was further demonstrated to be due to two-photon excitation by the increased anisotropy observed both from the steady-state and time-resolved data. Comparison of the intensities for one-photon and two-photon evanescent wave excitation revealed a smaller effective excited volume for two-photon excitation, indicating that the excited fluorophores are located closer to the interface with two-photon excitation. These results suggest that total internal reflectance can be combined with two- or multiphoton excitation for studies of surface absorption, immunoassays, or pattern photobleaching.
The detection of two-photon excited fluorescence is practically free from background caused by linear scattering because two-photon excited fluorescence occurs at a much shorter wavelength region than the excitation light. This property can be used to achieve ultrasensitive fluorescence detection. However, similar to linear scattering in the detection of one-photon excited fluorescence, the question arises whether background caused by non-linear scattering may limit the detection sensitivity of two-photon excited fluorescence. In this work, quantitative comparisons between two-photon induced scattering of liquid water and two-photon excited dye fluorescence in a standard epifluorescence geometry show that the relative scattering background is typically reduced by orders of magnitude in two-photon excitation as compared to single-photon excitation with confocal detection. Hyper-Rayleigh and hyper-Raman (3400 cm-1) cross sections of liquid water have been measured to be 8 x 10(-62) and 7 x 10(-63) cm4.s/photon, respectively, at 840 nm incident wavelength, with absolute values calibrated with respect to the known two-photon fluorescence excitation cross section of fluorescein.