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Video-rate hyperspectral two-photon fluorescence microscopy for in vivo imaging

机译:视频速率高光谱双光子荧光显微镜用于体内成像

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Fluorescence hyperspectral imaging is a powerful tool for in vivo biological studies. The ability to recover the full spectra of the fluorophores allows accurate classification of different structures and study of the dynamic behaviors during various biological processes. However, most existing methods require significant instrument modifications and/or suffer from image acquisition rates too low for compatibility with in vivo imaging. In the present work, a fast (up to 18 frames per second) hyperspectral two-photon fluorescence microscopy approach was demonstrated. Utilizing the beam-scanning hardware inherent in conventional multi-photon microscopy, the angle dependence of the generated fluorescence signal as a function beam's position allowed the system to probe of a different potion of the spectrum at every single scanning line. An iterative algorithm to classify the fluorophores recovered spectra with up to 2,400 channels using a custom high-speed 16-channel photon multiplier tube array. Several dynamic samples including live fluorescent labeled C. elegans were imaged at video rate. Fluorescence spectra recovered using no a priori spectral information agreed well with those obtained by fluorimetry. This system required minimal changes to most existing beam-scanning multi-photon fluorescence microscopes, already accessible in many research facilities.
机译:荧光高光谱成像是用于体内生物学研究的强大工具。恢复荧光团全光谱的能力允许对不同结构进行准确分类,并研究各种生物过程中的动力学行为。然而,大多数现有方法需要重大的仪器修改和/或图像采集速率太低而无法与体内成像兼容。在目前的工作中,展示了一种快速(每秒高达18帧)的高光谱双光子荧光显微镜方法。利用传统的多光子显微镜固有的光束扫描硬件,所产生的荧光信号的角度依赖性作为功能光束的位置,使系统可以在每条扫描线上探测光谱的不同部分。一种使用自定义高速16通道光子倍增管阵列的迭代算法,可对多达2400个通道的荧光团回收光谱进行分类。以视频速率对包括动态荧光标记的秀丽隐杆线虫在内的几个动态样品进行了成像。不使用先验光谱信息而回收的荧光光谱与通过荧光测定法获得的荧光光谱非常吻合。该系统需要对大多数现有的束扫描多光子荧光显微镜进行最小的改动,许多研究机构已经可以使用这种显微镜。

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