Temporally and spectrally resolved imaging microscopy of lanthanide chelates
Temporally and spectrally resolved imaging microscopy of lanthanide chelates
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DOI：
10.1016/s0006-3495(98)77930-5
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发表时间：
1998-05-01
影响因子：
3.4
通讯作者：
Jovin, TM
中科院分区：
文献类型：
--
作者：
Vereb, G;Jares-Erijman, E;Jovin, TM
在基于长寿命发光标记的荧光显微镜中，时间分辨率和光谱分辨率的结合由于抑制了散射光和短寿命自体荧光，大大提高了对比度和探测器的选择性。我们描述了一种将光谱和微秒时间分辨率与基于CCD相机的常规数字成像相结合的荧光显微镜的各种配置。微秒氙气闪光灯提供的高功率、宽光谱分布和微秒时间分辨率提供了最近开发的寿命在亚微秒到微秒范围内的荧光团的更高光度。在检测方面，门控微通道板增强器提供所需的时间分辨率和信号放大，光谱分辨率由双光栅色散光谱仪实现，并已应用于现场细胞化学样品和微室中极小体积元素的发光标记分析。另外引入偏振光学使得能够确定发射偏振；该参数反映了分子取向和相互关系的迁移率，从而反映了微环境的性质。采集的双重光谱和时间分辨率模式与空间、光谱和时间维度上的后验图像分析相辅相成，形成了一种非常灵活和通用的工具。我们使用了一种新开发的稀土螯合物Eu-DTPA-cs124来展示这些能力。这些化合物是时间分辨成像显微镜和通过荧光(发光)共振能量转移估计分子在显微镜中的接近程度和通过荧光去偏振估计分子旋转的良好标记物。我们描述了在显微镜下成像的稀土络合物晶体的光谱分布、偏振态和激发态寿命。
The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal, Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and relational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.