Annular Beam Shaping in Multiphoton Microscopy to Reduce Out-of-Focus Background

Johan Borglin, Danni Wang, N. Durr, D. Hanstorp, A. Ben-Yakar, M. Ericson
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引用次数: 4

Abstract

Despite the inherent spatial confinement of multiphoton processes that arises from focusing through an objective, the maximum imaging depth in conventional multiphoton microscopy is ultimately limited by noise from out-of-focus fluorescence. This is particularly evident when imaging beyond shallow depths in highly scattering tissue as increased laser powers are necessary. The out-of-focus signal originates from multiphoton processes taking place primarily at shallow depths and deteriorates contrast and limits imaging depth. In this paper, annular laser beams are explored as a concept to reduce this background signal in multiphoton microscopy. The approach is theoretically verified by data from simulations and proof of principle is demonstrated on a custom-built experimental multiphoton microscopy platform. Annular laser beams were created by adopting wavefront control using a spatial light modulator and implemented for imaging tissue phantoms simulating turbid media and human skin ex vivo. The signal-to-background ratios were calculated and compared to images acquired with a traditional, filled-aperture Gaussian beam. Experiments in tissue phantom show an improvement in signal-to-background ratio of about 30% when using annular beam illumination in comparison to Gaussian illumination at specific depths. When laser power is not the limiting factor, this approach is expected to provide even greater benefits.
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环形光束整形在多光子显微镜减少失焦背景
尽管通过物镜聚焦产生的多光子过程的固有空间限制,但传统多光子显微镜的最大成像深度最终受到失焦荧光噪声的限制。这是特别明显的,当成像超过浅深度的高散射组织,增加激光功率是必要的。失焦信号源于主要发生在浅深度的多光子过程,它降低了对比度并限制了成像深度。本文探讨了在多光子显微镜中,环形激光束作为一种减小背景信号的概念。该方法在理论上得到了仿真数据的验证,并在一个定制的实验多光子显微镜平台上进行了原理验证。利用空间光调制器采用波前控制产生环形激光束,实现了模拟浑浊介质和人体皮肤的组织幻象离体成像。计算了信号背景比,并与传统的填充孔径高斯光束获得的图像进行了比较。实验表明,在特定深度下,环形光束照明比高斯照明可提高30%左右的信本比。当激光功率不是限制因素时,这种方法有望提供更大的好处。
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