Axial high-resolution differential confocal microscopy

In this paper, an axial high-resolution differential confocal microscopy (AHDCM) is proposed to improve the axial resolution of confocal microscopy (CM). AHDCM divides the detection optical path of the confocal system into three paths: focal-plane detection imaging optical path, pre-focal detection imaging optical path and post-focal detection imaging optical path. The pre-focal and the post-focal detection imaging optical paths have the equal axial detector offset oriented in opposite direction. Subsequently, the axial resolution of the confocal system is improved by subtracting the sum of the pre-focal and post-focal detection signals from the twice focal-plane detection signal. Theoretical analyses and preliminary experiments indicate that, for an emitting wavelength of lambda = 405 nm and numerical aperture of NA = 0.8, the AHDCM's axial resolution improves by more than 35% compared with that of CM. Therefore, AHDCM is a practical and feasible new method for axial high-resolution microscopic imaging.