BackgroundHematoxylin and Eosin staining in paraffin embedded tissue (H&E staining in paraffin embedded tissue) is a widely used method in the incision dimensions measurement. Since H&E staining in paraffin embedded tissue requires complicated process and spends a lot of time, a direct measurement method based on a microscope is proposed to measure the incision dimensions, which greatly improves the efficiency.AimIn this paper, a direct measurement method using optical microscope and an indirect measurement method based on H&E staining in paraffin embedded tissue are presented and compared, so as to obtain a better method of measurement efficiency and accuracy.ApproachThe femtosecond laser is used to process chicken breast tissue, the directly measurement method based on a microscope and indirectly measurement method based on H&E staining are used to measure dimensions. Moreover, the laser confocal microscopy measurement (LCM) is also used to measure the incision depth.ResultsIt was found that the direct measurement method has high measurement efficiency and accuracy for incision dimensions measurement.ConclusionsOverall, the incision dimensions obtained by femtosecond laser processing can be measured through the direct measurement method instead of H&E staining in paraffin embedded tissue.