A 4-GHz frequency-domain fluorometer with internal microchannel plate photomultiplier cross-correlation

Klaus W. Berndt, Ignacy Gryczynski, Joseph R. Lakowicz

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

We have developed and tested a multifrequency phase/modulation fluorometer based on the Hamamatsu Model R2024U gatable microchannel plate photomultiplier (MCP-PMT), using internal MCP-PMT cross-correlation. This internal mixing is accomplished by biasing and modulating the gating mesh which is located 0.2 mm behind the photocathode. Near the photocathode center, no high-frequency photocurrent modulation was achieved. Within a circular area near the photocathode edge, however, the R2024U allows accurate phase shift and demodulation measurements up to at least 4.5 GHz, the frequency limit of our PMT-modulation amplifier. By mixing immediately after the photocathode, there is no decrease in the time resolution due to transit time spread, and the MCP has to process only low-frequency signals. This means no low-level high-frequency signal voltages have to be handled in this fluorometer, and the problems of RF shielding become much less critical. Also, the effective output impedance of the PMT has been increased, resulting in a 43-dB increase in the PMT output signal power. In principle, more MCPs could be built into the PMT, allowing an improved fluorescence detection limit. We have used the method of reference fluorophores in order to compensate for pronounced PMT color effects, a wavelength-dependent modulation, and a wavelength-dependent time shift. No color correction is required in the case of time-dependent depolarization. The performance of the instrument was verified by measurements of the intensity decay of perylene, which showed a single-exponential decay, and by measurements of the decay of tryptophan in water, which showed a double-exponential decay, as expected. Additionally, we measured a 231-ps decay time for rose bengal in a methanol:water mixture (50:50), and a 183-ps correlation time. This method of detection and mixing could be of particular usefulness in studies of time-dependent photon migration in tissues, which require high modulation frequencies, but do not require the detector to be free of color effects.

Original languageEnglish
Pages (from-to)131-137
Number of pages7
JournalAnalytical Biochemistry
Volume192
Issue number1
DOIs
Publication statusPublished - 1 Jan 1991

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