Multifocal multiphoton microscopy with adaptive optical correction
Multifocal multiphoton microscopy with adaptive optical correction
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DOI：
10.1117/12.2000188
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发表时间：
2013-02
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影响因子：
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通讯作者：
Simao Coelho;S. Poland;N. Krstajić;D. Li;J. Monypenny;R. Walker;D. Tyndall;T. Ng;R. Henderson-R.-Hend
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文献类型：
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作者：
Simao Coelho;S. Poland;N. Krstajić;D. Li;J. Monypenny;R. Walker;D. Tyndall;T. Ng;R. Henderson-R.-Hend
荧光寿命成像显微镜(FLiM)是一种成熟的方法，用于测量活细胞内的动态信号事件，包括检测蛋白质-蛋白质相互作用。与线性激发相比，由于瑞利散射和低吸收，红外光的光学穿透性得到了改善，在脑组织中提供了高达1 mm的成像深度，但当样品扭曲(像差)红外激发光束时，图像会显著退化。多光子时间相关单光子计数(TCSPC)薄膜是一种获得生物结构的功能、高分辨率图像的方法。为了获得良好的统计精度，TCSPC通常需要较长的采集时间。我们报道了一种名为MegaFLI的多焦点多光子显微镜(MMM)的发展。通过使用空间光调制器(SLM)的3D Gerchberg-Saxton(GS)算法执行的光束并行化，增加了与产生的小光束的数量成比例的TCSPC计数速率。采用加权3DGS算法来提高图像的均匀性。另一个好处是实现了灵活和自适应的光学校正。利用Zernike多项式进行的自适应光学被用来校正系统诱导的像差。在这里，我们展示了使用新型互补金属氧化物半导体(CMOS)1024像素单光子雪崩二极管(SPAD)阵列获得的吞吐量显著提高的结果，为实现真正的高通量薄膜开辟了道路。
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.