Vizsnyiczai Gaszton, Búzás András, Lakshmanrao Aekbote Badri, Fekete Tamás, Grexa István, Ormos Pál, Kelemen Lóránd
Institute of Biophysics, Biological Research Centre, Temesvári krt. 62, Szeged, 6726, Hungary.
Doctoral School of Physics, Faculty of Science and Informatics, University of Szeged, Dugonics square 13, Szeged, 6720, Hungary.
Biomed Opt Express. 2020 Jan 16;11(2):945-962. doi: 10.1364/BOE.379233. eCollection 2020 Feb 1.
Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution that is several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imaging of single cells is implemented by rotating them around an axis perpendicular to the optical axis by means of holographic optical tweezers. For this, the cells are indirectly trapped and manipulated with special microtools made with two-photon polymerization. The cell is firmly attached to the microtool and is precisely manipulated with 6 degrees of freedom. The total control over the cells' position allows for its multiview fluorescence imaging from arbitrarily selected directions. The image stacks obtained this way are combined into one 3D image array with a multiview image processing pipeline resulting in isotropic optical resolution that approaches the lateral diffraction limit. The presented tool and manipulation scheme can be readily applied in various microscope platforms.
由于显微镜光学系统的点扩散函数呈细长状，细胞的荧光观察通常受到轴向分辨率有限的影响。因此，三维成像的轴向分辨率比横向分辨率差几倍。解决这个问题的光学方法通常需要复杂的光学器件和极高的空间稳定性。消除各向异性分辨率的一种直接方法是融合从多个观察方向记录的图像，这主要通过整个样品的机械旋转来实现。在本文提出的方法中，单细胞的多视图成像是通过全息光镊围绕垂直于光轴的轴旋转细胞来实现的。为此，使用双光子聚合制作的特殊微工具间接捕获和操纵细胞。细胞牢固地附着在微工具上，并通过六个自由度进行精确操纵。对细胞位置的全面控制允许从任意选定的方向对其进行多视图荧光成像。通过这种方式获得的图像堆栈通过多视图图像处理管道组合成一个三维图像阵列，从而产生接近横向衍射极限的各向同性光学分辨率。所提出的工具和操纵方案可以很容易地应用于各种显微镜平台。
