Fluorescence Lifetime Measurement via a Radionuclide-Scintillation Light Source and Analog Cross Correlation

Abstract

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.

Digital cross-correlation technique for fluorescence-lifetime measurement using a radionuclide-scintillation excitation source

“…25 The compact optical configuration left no air gaps between components. The emission intensity of Pilot U is rated to be 65% that of anthracene and corresponds to approximately 10 000 photons per MeV of absorbed beta irradiation ͑NE Technologies͒.…”

“…25 Because the manufacturer's specifications for mixer local-oscillator ͑L͒ driving power, interport isolation, and bandwidth were determined with sinusoidal test waveforms, we selected four different mixers with slightly different characteristics in order to determine the one which was best suited for use with nanosecond pulses. Comparison experiments were performed with the air-gap optical configuration.…”

“…The spectral overlap of the light-source emission spectrum and the perylene excitation band are shown in Fig. We conducted a pulse-height analysis 25 to determine the actual number of photons that exit both the scintillator and the 400 nm excitation filter. Transmission through a 435 nm emission filter is also displayed and indicates the bandwidth incident on the fluorescence detector.…”

“…18 Stadelmann was the first to use TCSPC in conjunction with a radionuclide-scintillator excitation source to measure the lifetimes of organic fluorophores. 25 This approach fully exploited the random nature of the excitation source and allowed very accurate lifetime measurements. Hobbs and Hieftje demonstrated an improved beta-emitting version which also utilized TCSPC for lifetime analysis.…”

Photon-counting 1.0 GHz-phase-modulation fluorometer

“…20,[29][30][31]33 In this fashion, the SNR and the sensitivity are improved, as was done by the conventional cross correlation technique. [34][35][36] Resultantly, the precise phase (or amplitude) measurements for obtaining the fluorescence lifetime values are performed with success. 28 Nowadays, the heterodyne (homodyne) technique is introduced in the field of fluorescence lifetime imaging microscopy (FLIM), where a combination of a gain modulated image intensifier and a two-dimensional imaging camera is usually used.…”