Rodrigo José A, Soto Juan M, Alieva Tatiana
Universidad Complutense de Madrid, Facultad de Ciencias Físicas, Ciudad Universitaria s/n, Madrid 28040, Spain.
Biomed Opt Express. 2017 Nov 9;8(12):5507-5517. doi: 10.1364/BOE.8.005507. eCollection 2017 Dec 1.
The refractive index (RI) is an important optical characteristic that is often exploited in label-free microscopy for analysis of biological objects. A technique for 3D RI reconstruction of living cells has to be fast enough to capture the cell dynamics and preferably needs to be compatible with standard wide-field microscopes. To solve this challenging problem, we present a technique that provides fast measurement and processing of data required for real-time 3D visualization of the object RI. Specifically, the 3D RI is reconstructed from the measurement of bright-field intensity images, axially scanned by a high-speed focus tunable lens mounted in front of a sCMOS camera, by using a direct deconvolution approach designed for partially coherent light microscopy in the non-paraxial regime. Both the measurement system and the partially coherent illumination, that provides optical sectioning and speckle-noise suppression, enable compatibility with wide-field microscopes resulting in a competitive and affordable alternative to the current holographic laser microscopes. Our experimental demonstrations show video-rate 3D RI visualization of living bacteria both freely swimming and optically manipulated by using freestyle laser traps allowing for their trapping and transport along 3D trajectories. These results prove that is possible to conduct simultaneous 4D label-free quantitative imaging and optical manipulation of living cells, which is promising for the study of the cell biophysics and biology.
折射率（RI）是一种重要的光学特性，常用于无标记显微镜中对生物物体进行分析。一种用于活细胞三维折射率重建的技术必须足够快，以捕捉细胞动态，并且最好能与标准宽视场显微镜兼容。为了解决这个具有挑战性的问题，我们提出了一种技术，该技术能够快速测量和处理实时三维物体折射率可视化所需的数据。具体而言，通过安装在sCMOS相机前的高速聚焦可调透镜进行轴向扫描，利用为非傍轴区域的部分相干光显微镜设计的直接去卷积方法，从明场强度图像的测量中重建三维折射率。测量系统和提供光学切片和散斑噪声抑制的部分相干照明，都使得该技术与宽视场显微镜兼容，成为当前全息激光显微镜具有竞争力且经济实惠的替代方案。我们的实验演示表明，通过使用自由式激光阱对活细菌进行自由游动和光学操控，可实现视频速率的三维折射率可视化，从而允许对细菌进行捕获并沿三维轨迹运输。这些结果证明，对活细胞进行同时的四维无标记定量成像和光学操控是可行的，这对于细胞生物物理学和生物学的研究具有重要意义。
