Wen Chenyang, Ren Mindan, Feng Fu, Chen Wang, Chen Shih-Chi
Opt Lett. 2019 Sep 1;44(17):4343-4346. doi: 10.1364/OL.44.004343.
3-D two-photon excitation (TPE) microscopy has been a critical tool for biological study since its introduction. Yet, the speed is largely limited by its point detector, e.g., photomultiplier tube (PMT), which requires a point-scanning imaging sequence. In this Letter, we present a multi-focus compressive sensing (CS) method for 3-D and random-access TPE microscopy based on a digital micromirror device (DMD). This new platform combines CS with a unique holography-based DMD random-access scanner to enhance the imaging speed by three to five times for imaging arbitrarily selected regions in 3-D specimens without sacrificing the resolution. In the experiments, 1-20 randomly selected foci are generated by modulating the wavefront of a femtosecond laser via binary holography, where the combined intensity is recorded by a PMT. By exploiting CS algorithms, 3-D images at arbitrarily selected sites can be reconstructed. Simulations and imaging experiments on different samples have been performed to verify the principle and identify the optimal processing parameters, including the number of laser foci and sampling ratios. The results show that high-resolution images can be obtained by using a 25% sampling ratio and five foci. The new CS-based TPE imaging method may find important applications in biological studies, e.g., neuronal imaging and optogenetics.
自三维双光子激发（TPE）显微镜问世以来，它一直是生物学研究的关键工具。然而，其速度在很大程度上受限于点探测器，例如光电倍增管（PMT），这需要点扫描成像序列。在本信函中，我们提出了一种基于数字微镜器件（DMD）的用于三维随机访问TPE显微镜的多焦点压缩感知（CS）方法。这个新平台将CS与独特的基于全息术的DMD随机访问扫描仪相结合，在不牺牲分辨率的情况下，将三维样本中任意选定区域的成像速度提高了三到五倍。在实验中，通过二元全息术调制飞秒激光的波前产生1至20个随机选定的焦点，其合成强度由PMT记录。利用CS算法，可以重建任意选定位置的三维图像。已针对不同样本进行了模拟和成像实验，以验证原理并确定最佳处理参数，包括激光焦点数量和采样率。结果表明，使用25%的采样率和五个焦点可获得高分辨率图像。这种基于CS的新型TPE成像方法可能在生物学研究中找到重要应用，例如神经元成像和光遗传学。
