用于高效受激发射损耗显微镜荧光染料的光谱学原理。

Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores.

机构信息

Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium.

出版信息

J Am Chem Soc. 2010 Apr 14;132(14):5021-3. doi: 10.1021/ja100079w.

PMID:20307062

Abstract

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.

摘要

我们提出了一种用于鉴定受激发射损耗（STED）显微镜中优越染料的基本原理。我们比较了 pPDI 和 pTDI 两种染料，它们在单分子光谱学中表现出优异的光稳定性。令人惊讶的是，它们在 STED 显微镜中的光稳定性和性能有显著差异。虽然可以用优异的分辨率（35nm）可视化单个 pTDI 分子，但在类似条件下，pPDI 分子会迅速漂白。飞秒瞬态吸收测量证明，受激发射带和激发态吸收带之间的重叠是观察到的差异的主要原因。因此，评估激发态吸收带为染料选择提供了一种合理的方法，并确定了 STED 的最佳波长。