Sophie A Meredith, Yuka Kusunoki, Stephen D Evans, Kenichi Morigaki, Simon D Connell, Peter G Adams
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引用次数: 0
Abstract
It is important to understand the behaviors of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called "fluorescence quenching" occurs when fluorophores are present at high concentrations, but the mechanisms for quenching are debated. Here, we used a technique called "in-membrane electrophoresis" to generate concentration gradients of fluorophores within a supported lipid bilayer, across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behavior of three commonly used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD), and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). FLIM images provided evidence of quenching in regions where the fluorophores accumulated, but the degree of quenching varied between the different fluorophores. The relationship between quenching and concentration was quantified and the "critical radius for trap formation," representing the relative quenching strength, was calculated as 2.70, 2.02, and 1.14 nm, for BODIPY, TR, and NBD, respectively. The experimental data support the theory that quenching takes place via a "transfer-to-trap" mechanism which proposes, firstly, that excitation energy is transferred between fluorophores and may reach a "trap site," resulting in immediate energy dissipation, and, secondly, that trap sites are formed in a concentration-dependent manner. Some previous work suggested that quenching occurs only when fluorophores aggregate, or form long-lived dimers, but our data and this theory argue that traps may be "statistical pairs" of fluorophores that exist only transiently. Our findings should inspire future work to assess whether these traps can be charge-transfer states, excited-state dimers, or something else.
期刊介绍:
BJ publishes original articles, letters, and perspectives on important problems in modern biophysics. The papers should be written so as to be of interest to a broad community of biophysicists. BJ welcomes experimental studies that employ quantitative physical approaches for the study of biological systems, including or spanning scales from molecule to whole organism. Experimental studies of a purely descriptive or phenomenological nature, with no theoretical or mechanistic underpinning, are not appropriate for publication in BJ. Theoretical studies should offer new insights into the understanding ofexperimental results or suggest new experimentally testable hypotheses. Articles reporting significant methodological or technological advances, which have potential to open new areas of biophysical investigation, are also suitable for publication in BJ. Papers describing improvements in accuracy or speed of existing methods or extra detail within methods described previously are not suitable for BJ.