Video-Rate Bioluminescence Imaging of MMP-2 Figure 1. Bioluminescence imaging of GLase as a reporter protein to visualize proteins on the surface of mammalian cells. (A) Schematic representation of imaging principles by GLase bioluminescence. (B) Detectable area of exocytosis in a single mammalian cell using GLase bioluminescence imaging method. (C) Schematic representation of the secretion of GLase and the fusion protein of GLase from mammalian cells and its binding on the cell surface. (D) Schematic representation of expression vectors for GLase and MMP2-GLase. GLuc (pcDNA3-GLuc); GLase with the signal peptide sequence, MMP2-GLuc (pcDNA3-hMMP2-GLuc); human MMP2 preproprotein fused to GLase. (E) GLase activity in the conditioned medium of HeLa cells. HeLa cells were transfected with pcDNA3-GLuc or pcDNA3-hMMP2-GLuc and cultured for 24 hr followed by incubation with HBSS for 60 min at 37uC. The luminescence activity in the conditioned medium was determined using a luminometer. doi:10.1371/journal.pone.0025243.g001 the method of a video-rate bioluminescence imaging with the combination of EM-CCD camera and the secretory luciferase, Gaussia luciferase (GLase). As a result, this method allowed us to visualize exocytotic protein secretion with a time resolution of 30– 500 ms per image, and was applied to investigate the secretory dynamics of MMP-2 in a migrating HeLa cell. We successfully demonstrated the specific sites for both secretion and binding of MMP-2 on the cell surface. Results and Discussion Video-rate bioluminescence imaging of protein secretion from a single HeLa cell with an EM-CCD camera and Gaussia luciferase Previously, we visualized the exocytotic events of protein secretion and the protein targeting on the cell surface of CHO- K1 and PC12D cells using GLase [26]. The secreted GLase is the smallest luciferase (168 amino acid residues) and it is useful reporter protein for bioluminescence imaging over the cells (Figure 1C), but we could not distinguish between the secretory protein by exocytosis and the localized protein on the cell surface in real time due to low luminescence sensitivity of a photon counting camera and a CCD camera [26]. Prior to the visualization of MMP-2 secretion from a migrating HeLa cell, we established an improved bioluminescence video imaging system using a high-speed sensitive EM-CCD camera as a detector attached to a microscope. The EM-CCD camera showed very high quantum efficiency with over 90% and the luminescence video images were recorded at full spatial resolution allowing acquisition of 32 frames per second (with a minimum exposure time of 30.5 ms per image) on a hard disk. The performance of our improved imaging system with EM-CCD camera was evaluated using GLase secretion from HeLa cells. The cells transiently expressing GLase were cultured on a glass-bottom dish and were incubated with Hank’s Buffered Salt Solution (HBSS) containing luciferin (coelenterazine). With an exposure time of 30.5, 100, or 500 ms per image, the luminescence video images of GLase secretion from a single HeLa cell with a high numerical aperture 406 objective lens (NA 1.30) were recorded (Figure 2; Videos S1, S2, and S3). The luminescence video images with an exposure time of 500 ms per image clearly showed that GLase was released from specific sites on a HeLa cell and rapidly diffused to the outside of the cell (Figure 2B; Video S1). The luminescence intensities in each image could be used as an indicator for the amounts of secreted protein and the number of exocytotic sites. Thus, we could estimate time-dependent changes of GLase secretion from a single cell. The estimated values of luminescence intensities on the video image showed that the amounts of GLase PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25243