title: Feasibility of in vivo multichannel optical imaging of gene expression: experimental study in mice.
abstract: OBJECTIVE
  To develop and test a multichannel reflectance imaging system for small animals on the basis of a previously developed single-channel setup.
  
  
  METHODS
  The imaging system was composed of modular parts, including a light source, excitation filters, emission filters, and a charged-coupled device for recording images. On the basis of generated excitation and absorption spectra of green fluorescent protein (GFP), tricarbocyanine 5.5 (Cy5.5), and indocyanine green (ICG), filters were selected to allow spectral separation and optimize resultant recorded signal. The system was tested by using a combination of the fluorochromes to confirm spectral separation. In vivo tests were performed in nude mice with tumors that expressed cathepsin B, which could be evaluated by using a Cy5.5-based activatable probe and GFP. For each in vivo tumor type and channel, statistical analysis was performed on the basis of signal intensity in the region of interest.
  
  
  RESULTS
  The different fluorochromes were readily distinguished with the system; characteristics such as power were determined for all wavelengths. The system demonstrated a linear response for GFP, a monotonic response for Cy5.5 over a range of more than three orders of magnitude of concentration, and a more complex response for ICG. In vivo analysis demonstrated the ability to image GFP expression and cathepsin B expression separately in tumors: As expected, marked differences were observed in GFP-expression imaging between tumor types (1,363 arbitrary units [AU] +/- 236 [SD] vs 110 AU +/- 11 for GFP-positive and GFP-negative tumors, respectively; P <.001), whereas similar cathepsin B expression (1,070 AU +/- 285 vs 1,168 AU +/- 367; P >.5) was observed. Histologic analysis confirmed in vivo findings.
  
  
  CONCLUSIONS
  Imaging multiple gene expressions simultaneously in vivo by using optical imaging is feasible.
authors.affiliation: Center for Molecular Imaging Research, CNY149-5403, Department of Radiology, Massachusetts General Hospital, Bldg 149, 13th St, Charlestown, MA 02129, USA. mahmood@helix.mgh.harvard.edu
authors.forename: Umar
authors.initials: U
authors.lastname: Mahmood
authors.forename: Ching-Hsuan
authors.initials: CH
authors.lastname: Tung
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D000818:Animals; D002232:Carbocyanines; D002401:Cathepsin B / Q000032:analysis / Q000235:genetics*; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007208:Indocyanine Green; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000201:enzymology
chemical_list: C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002401:Cathepsin B; D007208:Indocyanine Green