Design of a laser-based autofocusing microscope for a sample with a transparent boundary layer

This study proposes a new type of autofocusing microscope. The microscope was combined with a polarized beam-splitting system and an image-processing program to overcome the complex signals generated when focusing on a transparent boundary layer. The polarized beam-splitting system divides a source laser beam into four parallel beams using a birefringent crystal. It also uses an axicon lens to focus individually on different positions of the sample surface when light is incident on the objective lens to observe multiple-spot defocusing information simultaneously. The transparent boundary layer exhibits intricate signals when a sensor detects laser spots. Therefore, an image-processing program is proposed to filter unnecessary signals for calculating the evaluation function and driving the motor, which enables a focused view of the test sample to be attained conveniently. To prove the feasibility of the proposed autofocusing microscope, a frame was developed using a simulation software for observing the initial characteristics of the microscope and for conducting a qualitative analysis. Moreover, the image-processing effect on the sample was tested to determine whether the aim of the study was achieved. The experimental results matched suitably with the simulation results, which indicates that the proposed autofocusing microscope can solve the problem caused by the transparent boundary layer and can achieve a high focusing accuracy within a short focusing time.