Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).
Elimination of autofluorescence in fluorescence correlation spectroscopy using the AzaDiOxaTriAngulenium (ADOTA) fluorophore in combination with time-correlated single-photon counting (TCSPC).
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DOI：
10.1007/s00216-013-6879-0
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发表时间：
2013-05
影响因子：
4.3
通讯作者：
Fudala, Rafal
中科院分区：
文献类型：
--
作者：
Rich, Ryan M.;Mummert, Mark;Gryczynski, Zygmunt;Borejdo, Julian;Sorensen, Thomas Just;Laursen, Bo W.;Foldes-Papp, Zeno;Gryczynski, Ignacy;Fudala, Rafal
关键词：
荧光相关光谱（FCS）是一种经常应用的技术，可以精确和灵敏地分析分子扩散和相互作用。然而，FCS用于体外或离体研究的潜力尚未完全实现，部分原因是源于自体荧光的伪影（固有组分的荧光和固定剂诱导的荧光）。在这里，我们提出了azadioxatriangulenium（ADOTA）染料作为这个问题的解决方案。ADOTA探针的寿命约为19.4 ns，比大多数自发荧光组分长得多。因此，它可以很容易地分离的时间相关单光子计数（TCSPC）方法。在这里，我们证明了抑制FCS中的自发荧光，通过使用ADOTA标记的Hybryonan大分子（HA）与罗丹明123添加到模拟扩散荧光背景成分。罗丹明123的发射光谱和衰减速率与通常的自发荧光源重叠，其扩散行为是众所周知的。我们表明，从罗丹明123的贡献可以消除时间门控或荧光寿命相关光谱（FLCS）。虽然ADOTA和时间门控的配对是从荧光成像中去除自发荧光的有效策略，但光子的损失导致FCS的错误浓度值。另一方面，FLCS消除了自发荧光而没有这样的误差。然后，我们表明，时间门控和FLCS都可以成功地使用ADOTA标记的HA来检测透明质酸酶的存在，在许多类型的癌症中已经观察到透明质酸酶的过度表达。
Fluorescence Correlation Spectroscopy (FCS) is a frequently applied technique that allows for precise and sensitive analyses of molecular diffusion and interactions. However, the potential of FCS for in vitro or ex vivo studies has not been fully realized due in part to artifacts originating from autofluorescence (fluorescence of inherent components and fixative-induced fluorescence). Here, we propose the azadioxatriangulenium (ADOTA) dye as a solution to this problem. The lifetime of the ADOTA probe, about 19.4 ns, is much longer than most components of autofluorescence. Thus, it can be easily separated by time correlated single photon counting (TCSPC) methods. Here, we demonstrate the suppression of autofluorescence in FCS by using ADOTA labeled Hyaluronan macromolecules (HAs) with Rhodamine 123 added to simulate diffusing fluorescent background components. The emission spectrum and decay rate of Rhodamine 123 overlap with the usual sources of autofluorescence, and its diffusion behavior is well known. We show that the contributions from Rhodamine 123 can be eliminated by time-gating or by fluorescence lifetime correlation spectroscopy (FLCS). While the pairing of ADOTA and time-gating is an effective strategy for the removal of autofluorescence from fluorescence imaging, the loss of photons leads to erroneous concentration values with FCS. On the other hand, FLCS eliminates autofluorescence without such errors. We then show that both time gating and FLCS may be used successfully with ADOTA-labeled HA to detect the presence of hyaluronidase, the over-expression of which has been observed in many types of cancer.
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影响因子：
5.6
作者：
Weidemann, T;Wachsmuth, M;Langowski, J
通讯作者：
Langowski, J
影响因子：
3.2
作者：
Schnell, SA;Staines, WA;Wessendorf, MW
通讯作者：
Wessendorf, MW
影响因子：
10.8
作者：
Fore, Samantha;Yuen, Yin;Huser, Thomas
通讯作者：
Huser, Thomas
影响因子：
4.3
作者：
Rich, Ryan M.;Stankowska, Dorota L.;Maliwal, Badri P.;Sorensen, Thomas Just;Laursen, Bo W.;Krishnamoorthy, Raghu R.;Gryczynski, Zygmunt;Borejdo, Julian;Gryczynski, Ignacy;Fudala, Rafal
通讯作者：
Fudala, Rafal
DOI：
10.1073/pnas.93.15.7832
发表时间：
1996-07-23
影响因子：
11.1
作者：
Liu, DC;Pearlman, E;Sy, MS
通讯作者：
Sy, MS