Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy

This folder contains all raw data underlying the results presented in a manuscript, submitted for publication to Methods and Applications in Fluorescence, and entitled: Photo-physical characterization of high triplet yield brominated fluoresceins by transient state (TRAST) spectroscopy Authored by: Baris Demirbayᵃ, Glib Baryshnikovᵇ, Martin Haraldssonᶜ, Joachim Piguetᵃ, Hans Ågrenᵈ, Jerker Widengrenᵃ,* ᵃRoyal Institute of Technology (KTH), Experimental Biomolecular Physics, Department of Applied Physics, Albanova University Center, SE-106 91, Stockholm, Sweden ᵇLaboratory of Organic Electronics, Department of Science and Technology, Linköping University, SE-60174, Norrköping, Sweden ᶜChemical Biology Consortium Sweden, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden ᵈDepartment of Physics and Astronomy, Uppsala University, SE-751 20 Uppsala, Sweden *Corresponding Author: Email: [e-mail] (mailto:[e-mail]) , Phone: [phone] The data files are grouped into the different techniques used to generate them, and refer to the figures/tables in the manuscript where the extracted results are presented. ABSTRACT Photo-induced dark transient states of fluorophores can pose a problem in fluorescence spectroscopy, but their typically long lifetimes can also make them highly environment sensitive. This opens for exploiting these states as microenvironmental read-out parameters in bio-molecular spectroscopy and imaging, and to explore fluorophores with prominent dark-state formation yields to be used in such studies. In this work, we analyzed the singlet-triplet transitions of fluorescein by transient state (TRAST) spectroscopy and compared them with those of three synthesized carboxy-fluorescein derivatives, with one, two or four bromines linked to the anthracence backbone. By this bromination, a prominent internal heavy atom (IHA) enhancement of the intersystem crossing (ISC) rates was found, and a corresponding external heavy atom (EHA) enhancement upon adding potassium iodide (KI) into the fluorophore solutions. Notably, increased KI concentrations still resulted in lowered triplet state buildup in the brominated fluorophores, due to the relatively lower increase induced in the ISC, than in the triplet decay. Moreover, KI had an antioxidative effect on the fluorophores, resulting in an overall fluorescence enhancement of the brominated fluorophores. The mechanisms behind the prominent IHA effect were further investigated by density functional theory calculations, which suggest that the ISC likely takes place to a higher triplet state, followed by relaxation to the lowest triplet state. By TRAST measurements, performed under biologically relevant conditions and analyzing how the average fluorescence intensity of fluorescent molecules varies with a systematically varied excitation modulation, dark state transitions within very high triplet yield (>90%) fluorophores can be directly analyzed, as well as IHA and EHA effects. These measurements, not possible by other techniques such as fluorescence correlation spectroscopy, opens for bio-sensing applications based on high triplet yield fluorophores, and for characterization of high triplet yield photodynamic therapy agents, and how they are influenced by IHA and EHA effects.本文件夹包含一篇已投稿至《荧光方法与应用》（Methods and Applications in Fluorescence）期刊的手稿中所呈现研究结果的全部原始数据，该手稿标题为：基于瞬态态（transient state, TRAST）光谱的高三重态产率溴代荧光素光物理特性表征。 作者：Baris Demirbayᵃ, Glib Baryshnikovᵇ, Martin Haraldssonᶜ, Joachim Piguetᵃ, Hans Ågrenᵈ, Jerker Widengrenᵃ,* ᵃ瑞典皇家理工学院（Royal Institute of Technology, KTH）应用物理系实验生物分子物理研究组，阿尔巴诺瓦大学中心，SE-106 91 斯德哥尔摩，瑞典 ᵇ林雪平大学（Linköping University）科学与技术系有机电子实验室，SE-60174 北雪平，瑞典 ᶜ瑞典卡罗林斯卡研究所（Karolinska Institute）医学生物化学与生物物理学系瑞典化学生物学联盟（Chemical Biology Consortium Sweden）生命科学实验室，SE-171 77 斯德哥尔摩，瑞典 ᵈ乌普萨拉大学（Uppsala University）物理与天文学系，SE-751 20 乌普萨拉，瑞典 *通讯作者：电子邮箱：[e-mail]（mailto:[e-mail]），电话：[phone] 数据文件按照生成所用的不同实验技术进行分组，并对应手稿中呈现提取结果的图表与表格。 **摘要** 荧光团（fluorophore）的光诱导暗瞬态态常会给荧光光谱检测带来困扰，但这类态通常较长的寿命也使其对环境变化极为敏感。这一特性使得我们可以将这些暗态用作生物分子光谱与成像中的微环境读出参数，并探索具有显著暗态形成产率的荧光团用于此类研究。本工作通过瞬态态（TRAST）光谱分析了荧光素的单重态-三重态跃迁，并将其与三种合成的羧基荧光素衍生物进行了对比，这三种衍生物的蒽骨架上分别连接了1个、2个或4个溴原子。研究发现，溴代作用可显著增强系间窜越（intersystem crossing, ISC）速率的内部重原子（internal heavy atom, IHA）效应，并且在向荧光素溶液中加入碘化钾（potassium iodide, KI）时，观察到了对应的外部重原子（external heavy atom, EHA）增强效应。值得注意的是，随着KI浓度升高，溴代荧光素的三重态构建量反而降低，这是因为KI对系间窜越的提升幅度相对弱于其对三重态衰减的促进作用。此外，KI对荧光团具有抗氧化作用，可整体提升溴代荧光素的荧光强度。我们进一步通过密度泛函理论（density functional theory, DFT）计算探究了显著内部重原子效应背后的机制，结果表明系间窜越大概率先发生至高能三重态，随后弛豫至最低能量三重态。 通过在生物相关条件下开展的TRAST测量，并分析荧光分子的平均荧光强度随系统性调控的激发调制方式的变化规律，我们可以直接分析三重态产率超过90%的高产率荧光团的暗态跃迁，以及内部重原子与外部重原子效应。这类测量是荧光相关光谱（fluorescence correlation spectroscopy, FCS）等其他技术无法实现的，其为基于高三重态产率荧光团的生物传感应用，以及高三重态产率光动力治疗剂的表征及其受内部重原子与外部重原子效应的影响研究提供了可行途径。