In this paper, GLUT4 vesicles are observed in real-time under TIRF microscopy and a new three-dimensional single particle tracking algorithm according to the unique features of TIRF is put forward.

• 利用TIRF显微镜动态实时地观测了GLUT4囊泡在脂肪细胞内的运动,并针对TIRF显微镜消散场成像的特点,提出了一种新的三维单粒子跟踪算法。