Fahrbach Florian O, Gurchenkov Vasily, Alessandri Kevin, Nassoy Pierre, Rohrbach Alexander
Laboratory for Bio- and Nano-Photonics, Department of Microsystems Engineering,University of Freiburg, Freiburg, Germany.
Opt Express. 2013 Jun 3;21(11):13824-39. doi: 10.1364/OE.21.013824.
In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5-10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3-5 relative to linear excitation with Gaussian beams. These finding arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the best image resolution, contrast at both a minimum amount of artifacts and signal degradation along the propagation of the beam into the sample.
在本研究中，我们表明可以成功地将光片显微镜、自重构贝塞尔光束和双光子荧光激发的优势结合起来，以改善在诸如癌细胞簇等大型散射介质中的成像。相对于用贝塞尔光束进行的线性激发，我们实现了轴向图像分辨率提高近两倍，对比度提高5至10倍。相对于用高斯光束进行的线性激发，光片穿透深度可增加3至5倍。这些发现来自实验和计算机模拟。此外，我们提供了这些结果是如何构成的理论描述。我们研究了球体簇和肿瘤多细胞球体在照明光束传播方向上图像质量的变化。结果表明，由脉冲近红外贝塞尔光束和双光子激发产生的光片在光束传播到样品中时，能提供最佳的图像分辨率、对比度，同时伪影和信号退化最少。
