NIR Fluorescence Imaging with SNAP-Tag Figure 3. Concentration dependent reaction of BG-800 with stable cells. 293-SNAPf (A), MDA-SNAPf (B) and SKOV-SNAPf (C) stable cells were reacted with different concentrations of BG-800. The fluorescence signals from the reaction were scanned on Odyssey Infrared Imaging System and quantified. TO-PRO-3 staining was used as the internal control. The insets represent scanned images of BG-800 (100 nM) stained stable cells (SNAPf) and control cells (Ctrl). RFU, relative fluorescence unit. doi:10.1371/journal.pone.0034003.g003 BG-800 was applied. As a comparison, the parental cell lines were reacted with the same concentrations of BG-800, and minimal signals were observed (Fig. 3A–C, and inset), lending additional support that the signal from stable cells is specific. protein IFP1.4 [8]. The resulting stable cell line 293-IFP was also compared with 293-SNAPf/BG-800. The ratio of 293-IFP signal to HEK293 signal after biliverdin treatment was 2.1, which is about 14 times lower than that of 293-SNAPf/BG-800. The combination of BG-800 with SNAPf-ADRb2 offers better signal to background ratio than other systems To compare BG-800 with the commercially available SNAP-tag substrate BG-782, 293-SNAPf and its parental cell line HEK293 were labeled with either BG-800 or BG-782. The signal intensity of BG-800 reaction with 293-SNAPf was approximately five-fold higher than that of BG-782 (data not shown). The ratio of 293- SNAPf signal to HEK293 signal is defined as the signal-to- background (S/B) ratio. The S/B ratio of BG-800 (S/B = 29) was about two-fold higher than that of BG-782 (S/B = 14) (Fig. 4). We also generated HEK293 cells stably expressing the NIR fluorescent Tumors expressing SNAPf-ADRb2 can be visualized by BG-800 The BG-800 substrate was evaluated in vivo in mouse models. Nude mice bearing SKOV-SNAPf tumors (Tm-S) on one side and SK-OV-3 tumors (Tm-C) on the other side were injected with 10 nmol of BG-800 through the tail vein. Whole mouse images were acquired at 24 h after imaging agent administration. Fig. 5A showed that SNAPf-ADRb2 expressing tumor could be visualized by BG-800 at 24 h post injection. The higher fluorescence signal in Tm-S was revealed more clearly by ex vivo imaging after tissue dissection (Fig. 5A, inset). The ratios of Tm-S/Tm-C and Tm-S/ PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34003