The conventional polarized light imaging system can observe sub-microscopic molecular order non-destructively, quantitatively and without labeling or staining. Recently, a more sophisticated version, Abrio™ imaging system, than the conventional polarized light imaging system, was developed. This advanced polarized light imaging system has simplified the process of birefringent imaging, higher sensitivity and accuracy irrespective of sample orientation. By performing time-lapse observations using the advanced polarized light microscopy, we visualized mitotic spindles, especially kinetochore microtubules, of HeLa cells. Moreover, we successfully visualized the detailed structure of several filament bundles, which possibly are components of the contractile ring. Here, we report the potential of advanced polarized light imaging systems for imaging mitotic HeLa cells and the new insights obtained during this microscopic study.