Figure 4. Comparison of signal to background (S/B) ratios of different systems. HEK293 cells expressing either SNAPf-ADRb2 or IFP1.4 were used. Both SNAPf-ADRb2 and IFP1.4 are under the control of the CMV promoter. SNAPf-ADRb2 expressing cells (293-SNAPf) were stained with BG-800 (SNAPf/BG-800) or BG-782 (SNAPf-BG-782), respectively. IFP1.4 expressing cells were incubated with biliverdin (IFP/BV). The excess substrate and biliverdin were washed away after reaction. The fluorescence signals of the cells were scanned on the culture plate without trypsinization and concentration. Signals were detected at their respective optimal wavelength (Ex/Em: 785/820 nm for BG-800 and BG-782; 685/720 nm for IFP1.4). The signals of HEK293 parental cells stained with the respective substrates or biliverdin were defined as background. doi:10.1371/journal.pone.0034003.g004 muscle were 3.3160.43 and 12.362.9, respectively. However, it was noticed that the background fluorescence signal was high. This high background signal might be caused by accumulation of BG-800 in the skin, as demonstrated by ex vivo imaging analysis (Fig. 5B). Other organs with high BG-800 accumulation included kidney, liver and lung. To assess whether the tumor signal was from the specific labeling of SNAPf-ADRb2 by BG-800, the tumor lysate was analyzed by gel electrophoresis. Fig. 5C showed that Tm-S lysate contained a product with the same size as the product from the reaction between BG-800 and SKOV-SNAPf cell lysate. In comparison, neither muscle nor SK-OV-3 tumor (Tm- C) contained this product. This product was also absent from other tissue lysates, including liver, lung and kidney (data not shown). Quenched BG substrates To assess whether the non-specific background signal could be reduced by using quenched substrates, CBG-800-QC1 was synthesized by conjugating IRDye 800CW and IRDye QC1to the benzyl and guanine groups, respectively. To decrease the adverse steric effect of the bulky IRDye QC1, a PEG linker was incorporated between IRDye QC1 and guanine. This version of quenched substrate with a PEG linker was designated as CBG- 800-PEG-QC1 (Fig. S1). The quenching efficiencies of CBG-800- QC1 and CBG-800-PEG-QC1 were estimated to be 97% and 94%, respectively. When incubated with excess purified SNAPf-EGF protein, the reaction signal from CBG-800-PEG-QC1 increased over time, and approached the level of BG-800 after 6 h (Note that unreacted BG-800 was not separated from the reaction mixture). In contrast the reaction signal from CBG-800-QC1 was only 21% of that of BG-800 after the same period of reaction (Fig. 6A). Analysis by gel electrophoresis demonstrated that although the intensities varied, the molecular weights of the reaction products from all BG substrates were the same (Fig. 6B). Reaction with NIR Fluorescence Imaging with SNAP-Tag Figure 5. In vivo imaging with BG-800 substrate. (A) Xenograft tumors were established using either SK-OV-3 parental cells as the control (Tm-C, left side) or SKOV-SNAPf stable cells (Tm-S, right side). The mice were imaged 24 h after i.v. injection of 10 nmol of BG-800 substrate. The tumors were indicated with arrows. The tumors were dissected and imaged ex vivo after whole animal imaging (lower left corner inset). (B) Tissue distribution of BG-800. Tissues were dissected 24 h after BG-800 administration and imaged. Note that the tissues were from a different mouse as shown in (A). Ht, Heart; Int, intestine; Kd, kidney; Lg, lung; Lv, liver; Ms, muscle; Sk, skin; Spl, spleen; Tm-C, SK-OV- 3 tumor; Tm-S, SKOV-SNAPf tumor. (C) Gel analysis of tissue lysates from Ms, Tm-C, Tm-S and SKOV-SNAPf cell lysate reacted with BG-800 (Cell-S). doi:10.1371/journal.pone.0034003.g005 SNAPf-ADRb2 expressing cells revealed that CBG-800-PEG- QC1 produced stronger signal than CBG-800-QC1. However signals from both CBG-800-PEG-QC1 and CBG-800-QC1 were much weaker compared to that of BG-800 (data not shown), indicating that a quencher on the guanine adversely affected the reactivity. As the reaction performance of CBG-800-PEG-QC1 was superior compared to BG-800- QC1, CBG-800-PEG-QC1 was used for in vivo animal imaging tests. Minimal background signal was observed for the quenched substrate even at the early stage post probe injection when the clearance had not occurred (Fig. S2), indicating that the quenching effect of the probe persevered in vivo. However, CBG-800-PEG-QC1 failed to detect the SNAPf- ADRb2 expressing tumors under these conditions. PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34003