Abstract
Abstract
Fluorescence microscopy and spectroscopy have been extensively used as indispensible tools in biophysical and biological research. In these applications, except intrinsically fluorescent proteins, nearly all the biomolecules are labeled with organic fluorophores, which serve as messengers of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule spectroscopy. Fluorescence photobleaching, which is interpreted as termination of emission of photons, is usually the factor hindering the application of organic dyes. The photobleaching quantum yield reflects the photostability of organic dyes, and the latter, along with brightness etc., is one of the most important criteria for a good fluorophore. Improving the photostability of organic dyes by designing the structure is always a difficult task for organic chemists. It requires a comprehensive understanding of the mechanism of the photobleaching behavior of fluorophores.
It is the aim of this work to understand the mechanisms of photobleaching behaviors of organic dyes, terrylene diimide (TDI) and amino-trioxatriangulenium dye (A3-TOTA+). Photobleaching is usually seen as permanent loss of fluorescence. In this work, we show that organic fluorophores can be converted into another chemical compound after photobleaching following excitation at the absorption maximum, and the new compound can be excited at another wavelength and fluoresce again. The restored fluorescence is blue-shifted from original emission for TDI, and red-shifted in the case of A3-TOTA+. The conversion efficiency for TDI is ~ 5%, deduced from 104 single molecule measurements. A simple and practical method is introduced to study the characteristics of the photoproducts at the ensemble level. Control experiments reveal that the reaction leading to photobleaching is oxygen related, but the composition of the photoproducts remains inconclusive, because of the superior stability and low efficiency of conversion of TDI. A3-TOTA+ is found to have similar conversion after photobleaching. Due to the relatively poor photostability, the conversion can be directly conducted in solution, and the composition of the photoproducts is determined by NMR and mass spectrometry. Upon illumination, A3-TOTA+ degrades in a step-wise manner by de-ethylation on the periphery. The unusual red-shifted fluorescence from the photoproducts is not as intense as the original emission, but the photostability is improved.
The acquired knowledge about photoconversion can stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism of A3-TOTA+ degradation can provide ideas for designing a homologue with improved photostability.
Fluorescence microscopy and spectroscopy have been extensively used as indispensible tools in biophysical and biological research. In these applications, except intrinsically fluorescent proteins, nearly all the biomolecules are labeled with organic fluorophores, which serve as messengers of conformational changes and dynamics. The photophysical properties of organic dyes directly determine the quality of the experiments. So the better understanding of the photophysical properties of organic dyes, the better we are able to design the experiments and interpret the data, especially in single molecule spectroscopy. Fluorescence photobleaching, which is interpreted as termination of emission of photons, is usually the factor hindering the application of organic dyes. The photobleaching quantum yield reflects the photostability of organic dyes, and the latter, along with brightness etc., is one of the most important criteria for a good fluorophore. Improving the photostability of organic dyes by designing the structure is always a difficult task for organic chemists. It requires a comprehensive understanding of the mechanism of the photobleaching behavior of fluorophores.
It is the aim of this work to understand the mechanisms of photobleaching behaviors of organic dyes, terrylene diimide (TDI) and amino-trioxatriangulenium dye (A3-TOTA+). Photobleaching is usually seen as permanent loss of fluorescence. In this work, we show that organic fluorophores can be converted into another chemical compound after photobleaching following excitation at the absorption maximum, and the new compound can be excited at another wavelength and fluoresce again. The restored fluorescence is blue-shifted from original emission for TDI, and red-shifted in the case of A3-TOTA+. The conversion efficiency for TDI is ~ 5%, deduced from 104 single molecule measurements. A simple and practical method is introduced to study the characteristics of the photoproducts at the ensemble level. Control experiments reveal that the reaction leading to photobleaching is oxygen related, but the composition of the photoproducts remains inconclusive, because of the superior stability and low efficiency of conversion of TDI. A3-TOTA+ is found to have similar conversion after photobleaching. Due to the relatively poor photostability, the conversion can be directly conducted in solution, and the composition of the photoproducts is determined by NMR and mass spectrometry. Upon illumination, A3-TOTA+ degrades in a step-wise manner by de-ethylation on the periphery. The unusual red-shifted fluorescence from the photoproducts is not as intense as the original emission, but the photostability is improved.
The acquired knowledge about photoconversion can stimulate new pathways in engineering and designing photoconvertible fluorophores, based on the reaction with oxygen or other chemicals. Besides, this results show that dyes that convert into other emissive species could give problems when interpreting single molecule FRET systems. The revealed mechanism of A3-TOTA+ degradation can provide ideas for designing a homologue with improved photostability.
| Originalsprog | Engelsk |
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| Forlag | Department of Chemistry, Faculty of Science, University of Copenhagen |
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| Antal sider | 113 |
| Status | Udgivet - 2014 |