Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag

PLoS One. 2012;7(3):e34003. doi: 10.1371/journal.pone.0034003. Epub 2012 Mar 30.


Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAP(f) is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADRβ2) fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAP(f)-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAP(f)-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

MeSH terms

  • Animals
  • Cell Line
  • DNA Modification Methylases / chemistry
  • DNA Modification Methylases / genetics
  • DNA Repair Enzymes / chemistry
  • DNA Repair Enzymes / genetics
  • Fluorescent Dyes / chemistry*
  • Humans
  • Mice
  • Mice, Nude
  • Molecular Imaging / methods*
  • Neoplasms / metabolism*
  • Receptors, Adrenergic, beta-2 / chemistry
  • Recombinant Fusion Proteins / chemistry
  • Signal-To-Noise Ratio
  • Spectroscopy, Near-Infrared / methods*
  • Staining and Labeling
  • Transfection
  • Transplantation, Heterologous
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / genetics


  • Fluorescent Dyes
  • Receptors, Adrenergic, beta-2
  • Recombinant Fusion Proteins
  • Tumor Suppressor Proteins
  • DNA Modification Methylases
  • MGMT protein, human
  • DNA Repair Enzymes