Preclinical whole body time domain fluorescence lifetime multiplexing of fluorescent proteins

• Published in: Journal of biomedical optics Vol. 19; no. 4; p. 046005
• Main Authors: Rice, William L, Kumar, Anand T. N
• Format: Journal Article
• Language: English
• Published: United States Society of Photo-Optical Instrumentation Engineers 01.04.2014
• Subjects: Animals; Basis functions; Cell Line; Decay; Female; Filled plastics; Fluorescence; Fluorescent Dyes - chemistry; Image Processing, Computer-Assisted; Luminescent Proteins - chemistry; Lymph; Mice; Mice, Nude; Multiplexing; Neoplasms, Experimental; Optical Imaging - methods; Proteins; Research Papers: Imaging; Time domain; Whole Body Imaging - methods; fluorescent proteins; lifetime multiplexing; autofluorescence; metastasis
• ISSN: 1083-3668; 1560-2281; 1560-2281
• DOI: 10.1117/1.JBO.19.4.046005

The application of time domain (TD) fluorescence lifetime multiplexing for the detection of fluorescent proteins (FPs) in whole animals, in the presence of a strong background tissue autofluorescence and excitation light leakage is discussed. Tissue autofluorescence (AF) exhibits a nonexponential temporal response, distinct from the mono-exponential decay of FPs. This allows a direct separation of FP fluorescence from AF using a dual basis function approach. We establish the detection limits of this approach using in vitro and in vivo measurements. We also demonstrate, using an experimental model of lymph node metastasis, that FP-AF lifetime multiplexing provides a greater than 30-fold improvement in contrast-to-background ratio compared with continuous wave data. In addition, we show that TD detection can simultaneously discriminate between up to three red shifted FPs placed under the skin of a nude mouse based on their distinct fluorescence lifetimes.