Abstract: For a microscopic imaging scene, an all-in-focus image of the observation object is needed. Because of the limitation of the depth of field of the camera and the typically uneven surface of the observation object, an all-in-focus image is obtained through one shot with relative difficulty. In this case, an alternative method for obtaining the all-in-focus image is usually used, which is to fuse several images focusing on different depths with the help of multi-focus image fusion technology. Multi-focus image fusion is an important branch in the field of computer vision. It aims to use image processing technology to fuse the clear regions of multiple images, focusing on different objects in the same scene, and finally to obtain an all-in-focus fusion result. With the breakthrough of machine learning theory represented by deep learning, the convolutional neural network is widely adopted in the field of multi-focus image fusion. However, most methods only focus on improving network structure and use the simple one-by-one serial fusion method, which reduces the efficiency of multiple image fusion. In addition, the defocus spread effect in the fusion process, which causes blurred artifacts in the areas near focus map boundaries, can severely affect the quality of fusion results. In the application of microscopic imaging analysis, we proposed a maximum spatial frequency in the feature map (MSFIFM) fusion strategy. By adding a post-processing module in the convolution neural network based on unsupervised learning, the redundant feature extraction process in the one-by-one serial fusion is avoided. Experiments demonstrate that this strategy can significantly improve the efficiency of multi-focus image fusion with multiple images. In addition, we presented a correction strategy that can effectively alleviate the effect of defocus spread on the fusion result under the condition of ensuring the efficiency of the algorithm fusion.