Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.
Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.
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DOI：
10.1007/s00216-012-6623-1
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发表时间：
2013-02
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.;Stankowska, Dorota L.;Maliwal, Badri P.;Sorensen, Thomas Just;Laursen, Bo W.;Krishnamoorthy, Raghu R.;Gryczynski, Zygmunt;Borejdo, Julian;Gryczynski, Ignacy;Fudala, Rafal
样品自发荧光（组织固有成分的荧光和固定剂诱导的荧光）是生物样品中分子过程的直接成像中的重要问题。组织中大量天然存在的荧光组分导致与用于组织标记的典型荧光染料的发射重叠的宽发射。此外，自发荧光的特征在于由多个组分组成的复杂荧光强度衰减，其寿命范围从亚纳秒到几纳秒。由于这些原因，探针的真实的荧光信号难以与不需要的自体荧光分离。在这里，我们提出了一种方法，用于减少自发荧光的问题，通过利用azadioxatriangulenium（ADOTA）染料的荧光寿命约为15 ns，远长于那些的大多数自发荧光的组件。具有如此长寿命的探针使我们能够使用时间选通强度成像来将靶向染料的信号与自体荧光分离。我们已经通过实验表明，通过丢弃在激发脉冲的前20 ns内检测到的光子，信号背景比提高了五倍。这种时间门控消除了超过96%的自发荧光。使用可变时间门的分析可以使得能够在没有来自背景的贡献的情况下定量测定结合的探针。
Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.
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影响因子：
3.6
作者：
Hammershoj, Peter;Sorensen, Thomas Just;Laursen, Bo W.
通讯作者：
Laursen, Bo W.
影响因子：
3.2
作者：
Schnell, SA;Staines, WA;Wessendorf, MW
通讯作者：
Wessendorf, MW
DOI：
10.1073/pnas.0630280100
发表时间：
2003-03-18
影响因子：
11.1
作者：
Haralampus-Grynaviski, NM;Lamb, LE;Simon, JD
通讯作者：
Simon, JD
影响因子：
3.5
作者：
König, K;Riemann, I
通讯作者：
Riemann, I
影响因子：
3.6
作者：
Laursen, Bo W.;Sorensen, Thomas J.
通讯作者：
Sorensen, Thomas J.