An electron microscope is one of the common used instruments to observe microorganisms. The microorganisms need be slicing, dyeing and fixing before observation. In the slicing process, a sampling position may be shifted. Additionally, the single layer image of the sliced microorganisms (hereafter called “samples”) only can provide partial information. Stacking all respective images of the samples will help for obtaining more information than the single layer image provided. Therefore, in this research, the method of image alignment is achieved by finding patterns of the images of the samples and aligning the images of that with the patterns to form a three-dimension image. Accordingly, the three-dimension image of the microorganisms provides many faces information to overcome the defect of the single layer image.