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Two-photon excitation fluorescence microscopy with a high depth of field using an axicon

机译：使用轴锥的高光场双光子激发荧光显微镜

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摘要

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.
机译：在常规的双光子激发荧光显微镜中，物镜的数值孔径决定了横向分辨率和景深。在某些情况下，如对活生物组织中分布的动态事件进行功能成像，则需要改进的时间分辨率。因此，必须使用具有高景深的光学器件同时对不同轴向位置的物体成像。利用常规的显微镜物镜，以横向分辨率为代价来增加景深。为了克服这个限制，我们在两光子激发荧光显微镜系统中加入了轴锥。测量表明，轴锥提供了超过一毫米的景深，而横向分辨率则保持在微米级。因此，已经显示了基于轴锥的双光子显微镜能够在对激光束进行单次2D扫描的同时，产生样品的高分辨率投影图像，同时保持典型的双光子显微镜具有更高的组织穿透力。

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