A Parallel Multiharmonic Frequency-Domain Fluorometer for Measuring Excited-State Decay Kinetics Following One-, Two-, or Three-Photon Excitation

Abstract

We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-locked, pulse-picked femto-second Ti-sapphire laser as the excitation source for the determination of one-, two-, or three-photon excited time-resolved fluorescence anisotropy and intensity decay kinetics. In operation, the new instrument can provide a complete frequency-domain data set at 100 modulation frequencies in less than 1 min. The new instrument exhibits 5-10-ps measurement precision and it can rapidly and accurately recover complex excited-state fluorescence anisotropy and intensity decay kinetics under one-, two-, or three-photon excitation for dilute or optically dense samples that exhibit single or multiexponential decay kinetics. This latter aspect of the instrument is demonstrated by successfully determining the excited-state intensity decay kinetics for a dilute aqueous solution of rhodamine 6G dissolved in a high concentration of bromocresol green. This approach is extended by determining the excited-state fluorescence intensity decay kinetics of dilute fluorescein directly in undiluted, whole blood as a function of pH under two-photon excitation conditions. The high-speed capabilities of the new instrument are exploited by performing two-photon excited fluorescence anisotropy decay experiments on the fly for site-selectively labeled bovine serum albumin as it undergoes enzymatic digestion by trypsin.

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

“…The concentration dependent quenching follows a Stern-Volmer model of bimolecular quenching (29,30). The relationship between the observed fluorescent lifetime, f , and the quencher concentration, [Q], is given by (15) where k q is the bimolecular rate constant for quenching, [Q] is the concentration of quencher, and f o is the fluorescent lifetime in the absence of quencher.…”

“…These wavelengths were chosen to match the typical spectral ranges of fluorophores commonly used in biological experiments, particularly those utilizing wild type and mutant forms of the green fluorescent protein (GFP). We used rhodamine 6G as a reference because of extensive prior work indicating that the dye has a single-component lifetime (29,30,33) and a linear Stern-Volmer behavior (29,30). We encountered difficulties with fluorescein standard solutions because of lifetime inconsistencies attributed to photobleaching artifacts.…”

“…[3.90 ns(30)] and Magde et al[3.99 ns,(33)], and in a microscope by Buist et al[3.9 ns, (36)]. In methanol, we observed excellent agreement with Magde et al (4.13 ns), although both numbers (4.13 and 4.16 ns) were higher than the 3.7 ns obtained by extrapolation of more concentrated solutions(37), and the maximum value of 4.0 ns reported by Gumy et al(38).…”

The Concentration-Dependent Distribution of Tris(4,7'-diphenyl-1,10'-phenanthroline) Ruthenium (II) within Sol-Gel-Derived Thin Films

“…[1][2][3][4][5][6][7] Over the past decade, sol-gel-processed thin films have been used in concert with a number of Ru(II) tris α-diimine dopants to produce luminescence-based sensors for O 2 quantification, with potential clinical, environmental, and process control applications. [8][9][10][11] Particularly promising among the Ru(II) diimine series is tris(4,7'-diphenyl-1,10'-phenanthroline) Ru(II) ([Ru(dpp) 3 ] 2+ ) which exhibits a high luminescence quantum yield, a long-lived excited-state luminescence lifetime, good photostability, large emission Stokes shift, and high molar absorptivity in the blue-green spectral region. [12][13][14][15][16][17] The photophysics and photochemistry of Ru(II) diimine quenching by O 2 is also well understood.…”

Quantitative Imaging in the Laboratory: Fast Kinetics and Fluorescence Quenching

“…Quenching studies have been used to control observed fluorescence lifetimes (28,29), in fundamental studies (30), and to assist in the resolution of mixtures (31). Quenching has been used to assay for hydrolyzing enzymes (32) and in biophysical studies to obtain information about the environment in which a fluorophore is located in a protein (33).…”