© 2003 The Royal Microscopical Society Journal of Microscopy, Vol. 209, Pt 3 March 2003, pp. 223 – 227 Received 10 August 2002; accepted 25 October 2002 Blackwell Publishing Ltd. Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy D.-M. YANG*, C.-C. HUANG†, H.-Y. LIN‡, D.-P. TSAI‡, L.-S. KAO† , §, C.-W. CHI* & C.-C. LIN¶ *Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, China †Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic of China ‡Department of Physics, National Taiwan University, Taipei, Taiwan, Republic of China §Department of Life Science, National Yang-Ming University, Taipei, Taiwan, Republic of China ¶Department of Life Sciences, Chung Shan Medical University, no. 110, Sec. 1, Chien-Kuo N. Road, Taichung 402, Taiwan, Republic of China Key words. Enhanced green fluorescent protein, exocytosis, fusion, membrane, Rab3, total internal reflection fluorescence microscopy, transfection, vesicle. Summary Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over-expression of enhanced green fluorescent protein-tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge-coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K + are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell. Introduction Amperometric and membrane capacitance measurements have been used to monitor exocytotic events during hormone and neurotransmitter release (Chow et al., 1992; Neher, 1998; Yang & Kao, 2001). However, exocytotic events are difficult to observe directly because of the limitations of spatial and temporal resolution of conventional microscopy. Recently, total internal reflection fluorescence microscopy (TIRFM) was used to detect fusion events near the cell surface (Neher, 1998). The advantages of TIRFM are high spatial and temporal resolution of the image, and low photo-damage to the cells (Betz & Angleson, 1998; Neher, 1998). Based on the advanced developments of the ultrahigh numerical aperture (NA) objective lens, the cur- rent TIRFM can be used to precisely observe dynamic events occurring at the sample surface within the near-field region. The observation region is confined to a certain penetration depth which depends upon the refraction index of the sample and the incident angle of light (Axelrod, 1989). Rab3A is member of a small G protein family expressed in neuronal and secretory cells, and is thought to be involved in regulated exocytosis. Involvement of Rab3A protein in the fusion between secretory vesicles and plasma membrane has been proposed by Holz et al. (1994), Johannes et al. (1994) and Geppert et al. (1994). Burns et al. (1998) and Chung et al. (1999) further suggest that Rab3A is involved in the docking of vesicles with plasma membrane and recycling of secretory vesicles rather than the vesicle–plasma membrane fusion. To illustrate the key role of Rab3A, we used Rab3A fused with enhanced green fluorescent protein (EGFP), and overexpressed in PC12 cells to give bright and stable fluorescence for the direct observation of Rab3A using TIRFM. The dynamic inter- actions of the Rab3A-associated vesicles are then studied. Materials and methods Total internal reflection fluorescence microscopy Our TIRFM, consists of an inverted epi-fluorescence micro- scope (IX-70; Olympus) equipped with a high numerical Received 10 August 2002; accepted 25 October 2002 Correspondence: Chung-Chih Lin. Tel.: 886 4 2472 0022 (ext. 1804); fax: 886 2 2823 4898; E-mail: bmlcgcsmc@hotmail.com