Adaptive aberration correction in a confocal microscope

The main advantage of confocal microscopes over their conventional counterparts is their ability to optically “section” thick specimens; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful in biological applications. However, it is well known that the resolution and optical sectioning ability can be severely degraded by system or specimen-induced aberrations. The use of high aperture lenses further exacerbates the problem. Moreover, aberrations can considerably reduce the number of photons that reach the detector, leading to lower contrast. It is rather unfortunate, therefore, that in practical microscopy, aberration-free confocal imaging is rarely achieved. Adaptive optics systems, which have been used widely to correct aberrations in astronomy, offer a solution here but also present new challenges. The optical system and the source of aberrations in a confocal microscope are considerably different and require a novel approach to wavefront sensing. This method, based upon direct measurement of Zernike aberration modes, also exhibits an axial selectivity similar to that of a confocal microscope. We demonstrate an adaptive confocal fluorescence microscope incorporating this modal sensor together with a deformable membrane mirror for aberration correction. Aberration corrected images of biological specimens show considerable improvement in contrast and apparent restoration of axial resolution.

共焦显微镜（confocal microscope）相较于传统光学显微镜的核心优势，在于其可对厚样品进行光学切片（optical sectioning）；通过获取的薄图像切片，能够重构三维图像，这一特性在生物应用中尤为实用。但众所周知，系统或样品引入的像差（aberration）会严重降低其分辨率与光学切片能力，而使用大孔径透镜（high aperture lens）会进一步加剧这一问题。此外，像差会大幅减少到达探测器（detector）的光子数量，导致对比度（contrast）下降。因此在实际显微成像中，几乎无法实现无像差的共焦成像，这一点颇为遗憾。此前广泛用于天文学像差校正的自适应光学系统（adaptive optics system），为该问题提供了解决方案，但也带来了新的挑战。共焦显微镜的光学系统与像差来源与天文学场景存在显著差异，因此需要全新的波前传感（wavefront sensing）方案。本文提出的方法基于直接测量泽尼克像差模式（Zernike aberration mode），同时具备与共焦显微镜类似的轴向选择性（axial selectivity）。我们搭建了集成该模态传感器与可变形膜镜（deformable membrane mirror）的自适应共焦荧光显微镜，用于像差校正。经像差校正后的生物样品成像结果显示，图像对比度显著提升，轴向分辨率也得到了明显恢复。