Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation
Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation
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DOI：
10.1016/s0006-3495(99)77065-7
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发表时间：
1999-10-01
影响因子：
3.4
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Schwille, P;Haupts, U;Webb, WW
荧光探针的多光子激发（MPE）已成为一个有吸引力的替代在激光扫描显微镜的生物应用，因为在活体组织的光谱测量中遇到的许多问题，如光散射，自发荧光，和光损伤可以减少。本研究探讨了双光子激发（2 PE）与共焦单光子激发（1 PE）的荧光相关光谱（FCS）的细胞内应用的特点进行比较。FCS是测量分子浓度、迁移率参数、化学动力学和荧光物理学的一种有吸引力的方法。几个FCS在哺乳动物和植物细胞中的应用进行了概述，以说明1 PE和2 PE的能力。分析了组织中定量FCS所需的荧光团的光物理性质。在活细胞和细胞膜上的测量是可行的，具有合理的信噪比，即使与荧光团浓度低至单分子水平的采样体积。分子迁移率可以在很宽的特征时间常数范围内测量，从10（-3）到10（3）ms。虽然这两种激发方法都适用于薄制剂中的细胞内FCS，但2 PE可以显著改善混浊制剂（如植物细胞和组织中的深层细胞）中的信号质量。在相当的信号水平，2 PE最大限度地减少光漂白在空间限制性细胞室，从而保持长期的信号采集。
Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from similar to 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.