PHOTON FLUORESCENCE SPECT & DYNAMICS OF ENHANCED GREEN FLUORESCENT PROTEIN

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One-photon fluorescence correlation spectroscopy (FCS) has been conducted on a number of fluorescent dyes for photobleaching dynamics characterization has been studied experimentally. Photobleaching quantum yield (?b) of rhodamine green, cy2, and fluorescein, following one-photon excitation ( 100-fs pulses and CW), has been characterized. While rhodamine green did not bleach under pulsed excitation, fluorescein underwent photobleaching (?b=8.4x10-6 and 1.0x10-5) following the CW- and pulsed-excitation; respectively. Similar measurements has been carried out on cy3, allophycocyanin, B-phycoerythrin, R-phycoerythrin, R-phycocyanin and rose bengal using CW-one-photon FCS. Comparison was made between the measured ?b in this work and others whenever possible. For example, ?b~7.0x10-5 of R-phycoerythrin is in a good agreement with Whiet and Stryer (1998) using a different technique (1.1x10-5). While the bleaching of cy3 was independent of the singlet oxygen, no photobleaching was obser ved in de-oxygenated rose bengal suggesting the involvement of triplet state and singlet oxygen in photobleaching dynamics. Preliminary measurements on mutant green fluorescent protein (EGFP) as a function of excitation wavelength (405 and 488 nm) as well as pH-value (5.8 and 11.0) of the buffer were also carried out. The triplet state lifetime (?T) and the intersystem crossing rate (kISC) of those fluorophores were also analyzed using the intensity-dependent FCS results.