Video-Rate Bioluminescence Imaging of MMP-2 Figure 2. Video-rate bioluminescence imaging of GLase secretion from a single HeLa cell using an EM-CCD camera. HeLa cells were transfected with pcDNA3-GLuc and cultured for 24 h. (A) Bright-field image of a single cell. Objective lens; 406. (B) Bioluminescence image of GLase secretion obtained from all area of bright-field image (A). Luminescence signals were recorded with an exposure time of 500 ms per image for 75 s (Video image is in Video S1). Yellow arrows in the images at 0.5 s indicate the luminescence spots in three spots 1–3. (C) Time-dependent changes of the average (red line) and maximum (green line) of luminescence intensities from GLase secretion. The arrows with numbers of 1–3 indicate the luminescence positions, shown as yellow arrows at 0.5 s in (B). (D) Magnified luminescence image of GLase secretion obtained from the area of yellow square in (A). Luminescence signals were acquired with an exposure time of 30.5 ms per image for 4.7 s (Video image is in Video S3). doi:10.1371/journal.pone.0025243.g002 secreted from a cell fluctuated in a short time and the secretion of GLase varied in different sites on the cell (Figure 2C; Video S1). Luminescence video images with the shorter exposure times of 30.5 and 100 ms showed smaller luminescence spots could be observed and these small spots might be individual exocytotic events of GLase (Figure 2D; Video S2 and S3). In our previous imaging system using a photon-counting camera with a long exposure time for 1 min, the intercellular weak luminescence signals were detected after 10 min by incubating with coelenterazine [26]. This result suggested that coelenterazine was slowly incorporated into a cell by endocytosis and this weak luminescence signals were explained by the luminescence reaction of GLase with coelenterazine. On the other hand, the present imaging study using a high-speed sensitive EM-CCD camera was performed with shorter exposure times within 500 ms and the luminescence images of secreted GLase were obtained within 15 min. In our imaging conditions, the diffusive luminescence signals of secreted GLase from a HeLa cell were observed without any significant increase in background luminescence signals in the cells (Figure 2B and D, and Videos S1, S2, and S3). Thus, the time resolution and the detection sensitivity of luminescence signals were significantly improved, accompanying high signal-to-noise ratio of luminescence signals of secreted GLase for detection. Expression and characterization of the fused protein of MMP-2 to GLase in HeLa cells To express the fusion protein of MMP-2 with GLase in HeLa cells, the expression vector of MMP2-GLase (Figure 1D) was constructed and was transfected into HeLa cells, which is known to express endogenous MMP-2 [27,28]. The luminescence activity of MMP2-GLase secreted into the conditioned medium was determined with a luminometer and the luminescence intensity of MMP2-GLase for bioluminescence video imaging analysis was observed (Figure 1E). The luminescence intensity of MMP2- PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25243