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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.

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics.

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium-sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.