PubMed · 11687397
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P T Tran, F Chang. 2001. Transmitted light fluorescence microscopy revisited.. https://doi.org/10.2307/1543340
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We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.
The solvatochromic behavior of the green fluorescence protein (GFP) chromophore (p-hydroxybenzylideneimidazolone, p-HBDI) and its derivatives (p-methoxybenzylideneimidazolone, p-MeOBDI, and N-methyl-p-hydroxybenzylideneimidazolonium iodide, p-HBDIMe+) was studied using UV-vis-absorption spectroscopy in a wide array of solvents. The relative contribution of specific (polarity) vs nonspecific (hydrogen-bonding) solvation to the absorbance spectra was studied. On the basis of these data, we discuss the nature of the absorption peak of the protonated and deprotonated forms of the wild-type GFP.
Mitochondrial dysfunction may play an important role in the pathogenic mechanism of Huntington's disease (HD). However, the exact mechanism by which mutated huntingtin could cause bioenergetic dysfunction is still unknown. We have constructed a stable inducible yeast model of HD by expressing a human huntingtin fragment containing a mutant polyglutamine tract of 103Q fused to green fluorescent protein (GFP), and a control expressing a wild-type 25Q domain fused to GFP in a wild-type strain. We showed that in yeast cells expressing 103Q, cell respiration was progressively reduced after 4-6 h of induction with galactose, down to 50% of the control after 10 h of induction. The cell respiration defect results from an alteration in the function and amount of mitochondrial respiratory chain complex II+III, in congruency to data obtained from postmortem brain of HD patients and from toxin models. In our model, the production of reactive oxygen species (ROS) is significantly enhanced in cells expressing 103Q. Quenching of ROS with resveratrol partially prevents the cell respiration defect. Mitochondrial morphology and distribution were also altered in cells expressing 103Q, probably resulting from the interaction of aggregates with portions of the mitochondrial web and from a progressive disruption of the actin cytoskeleton. We propose a mechanism for mitochondrial dysfunction in our yeast model of HD in which the interactions of misfolded/aggregated polyglutamine domains with the mitochondrial and actin networks lead to disturbances in mitochondrial distribution and function and to increase in ROS production. Oxidative damage could preferentially affect the stability and function of enzymes containing iron-sulfur clusters such as complexes II and III. Our yeast model represents a very useful paradigm to study mitochondrial physiology alterations in the pathogenic mechanism of HD.