A 10-GHz frequency-domain fluorometer

We describe the design and performance of a 10-GHz harmonic-content frequency-domain fluorometer. The modulated excitation is provided by the harmonic content of a train of ps pulses. High-speed and/or high-frequency detection was attained with a triode-type microchannel plate photomultiplier tube (MCP PMT) from Hamamatsu, R-2566-6, with 6 μm channels. To minimize the cost of the electronic components, and to minimize the noise due to these components, the detection circuits consists of two frequency ranges, 10 MHz-2 GHz and 2-10 GHz. The upper frequency limit of 10 GHz is determined by the current MCP PMT, so the usual configuration includes a low-noise 2-10-GHz amplifier. This amplifier is easily replaced with a 2-18-GHz amplifier to allow operation to 18 GHz and the use of faster PMTs, should they become available in the future. Measurement of known optical delays demonstrates the accuracy of the instrument. For instance, a 1.69 ps optical delay was measured as 1.7±0.4 ps from 0.5 to 10 GHz, and 1.7±0.2 ps from 2 to 8 GHz, where the uncertainty indicates the maximum deviation from the expected value. The data were shown to be free of systematic errors by measurements on fluorophores with single exponential decays, with decay times ranging from 61 ps to 1.24 ns. Measurement of anisotropy decays with correlation times of 24 ps are shown and it is predicted that correlation times as short as 1 ps could be measured with this instrument. And finally, the sensitivity of the instrumentation was demonstrated by measurements of the very weak intrinsic tryptophan emission of deoxyhemoglobin, which displays decay times ranging from 2 to 820 ps.