Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.
Semi-autonomous real-time programmable fluorescence lifetime segmentation with a digital micromirror device.
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DOI：
10.1364/oe.26.031055
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发表时间：
2018-11
期刊：
影响因子：
3.8
通讯作者：
J. Aluko;Camille Perrin;V. Devauges;J. Nedbal;S. Poland;D. Matthews;J. Whittaker;S. Ameer-Beg
中科院分区：
文献类型：
--
作者：
J. Aluko;Camille Perrin;V. Devauges;J. Nedbal;S. Poland;D. Matthews;J. Whittaker;S. Ameer-Beg
时间相关单光子计数（TCSPC）是在生物测定中执行寿命光谱的金标准。使用激光扫描显微镜的传统荧光寿命成像（FLIM）由于点扫描视场中的所有像素而固有地慢。使用微通道板的TCSPC光谱学的宽场实现受益于以时间分辨率为代价的特别快的采集时间，并且从根本上受到光子计数率的限制。在这里，我们介绍了可编程寿命成像（PLI），结合了使用全内反射激发的宽视场成像的优点，采用最先进的TCSPC检测器技术，以面向对象的方式使用数字微镜器件（DMD）进行准确的寿命测定。荧光发射被投射到DMD上，以便于从视场中的各个对象顺序分割荧光，从而允许图像采集和测定的荧光寿命确定。PLI的灵敏度通过手动分割来自固定细胞测定的荧光来证明。我们还演示了PLI的自动化实现，使用相机作为反馈机制来分割成像视场中感兴趣的发射对象所产生的荧光，突出了仅在存在有价值信息的区域中进行测量的优势。因此，与激光扫描实现相比，PLI能够将荧光寿命数据的采集时间减少至少一个数量级。
Time-correlated single-photon counting (TCSPC) is the gold standard for performing lifetime spectroscopy in biological assays. Traditional fluorescence lifetime imaging (FLIM) using laser scanning microscopes are inherently slow due to point scanning all pixels in the field-of-view. Wide-field implementations of TCSPC spectroscopy using microchannel plates benefit from particularly fast acquisition times at the expense of temporal resolution, and are fundamentally limited by photon counting rates. Here, we introduce programmable lifetime imaging (PLI), combining the advantages of wide-field imaging using total internal reflection excitation with state-of-the-art TCSPC detector technology for accurate lifetime determination in an object-oriented manner using a digital micromirror device (DMD). The fluorescent emission is projected onto the DMD to facilitate the sequential segmentation of fluorescence from individual objects in the field-of-view, allowing for both image acquisition and fluorescence lifetime determination of the assay. The sensitivity of PLI is demonstrated by manually segmenting fluorescence from fixed cell assays. We also demonstrate an automated implementation of PLI, using a camera as a feedback mechanism to segment fluorescence produced by emitting objects of interest in the imaging field-of-view, highlighting the advantages of measurement only in areas where valuable information exists. As a result, PLI is able to reduce acquisition time of fluorescence lifetime data by at least an order of magnitude compared to laser scanning implementations.