A 4-GHz frequency-domain fluorometer with internal microchannel plate photomultiplier cross-correlation.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Berndt
K W
KW
University of Maryland School of Medicine, Department of Biological Chemistry, Baltimore 21201.
Gryczynski
I
I
Lakowicz
J R
JR
D005453:Fluorescence; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D014364:Tryptophan / Q000737:chemistry; D014867:Water / Q000737:chemistry
D005456:Fluorescent Dyes; D014867:Water; D014364:Tryptophan