de Groot Mattijs, Evans Conor L, de Boer Johannes F
Institute for Lasers, Life and Biophotonics Amsterdam, Department of Physics and Astronomy, VU University Amsterdam, de Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
Opt Express. 2012 Jul 2;20(14):15253-62. doi: 10.1364/OE.20.015253.
We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan.
我们提出了一种用于高分辨率三维荧光成像的新方法。与光束扫描共聚焦显微镜不同，在光束扫描共聚焦显微镜中，激光焦点必须在横向和轴向上进行扫描以采集一个体积的数据，而我们无需深度扫描就能获得深度信息。在这种方法中，发射的荧光沿向后方向收集，并通过一个相位板，该相位板将深度信息编码到光谱分辨干涉图案的相位中。我们证明，解码此相位信息可在500 µm的景深范围内实现优于4 µm的深度定位精度。在具有小得多的景深的高数值孔径配置中，可以实现几十纳米的定位精度。这种方法非常适合微型内窥镜，因为内窥镜尖端的空间限制使得深度扫描变得困难。我们通过从单次二维扫描构建离体小鼠心脏组织微血管的三维体积，展示了对复杂生物样品进行三维可视化的潜力。
