Discrimination and Quantification of Spectrally Similar Near-Infrared Probes by Time-Domain (TD) Optical Imaging in Acute Myeloid Leukemia Mouse Models.

The use of whole-body optical imaging in the near-infrared (NIR) spectrum (650–1100 nm) employing fluorescently labelled reagents recognising cell-specific biomarkers of leukemia has become a standard modality in preclinical models of the human disease. A particular challenge is represented by leukemic infiltrates in liver and spleen, organs with high optical absorbance. While there are increasingly impressive arrays of fluorescently labelled biomolecules available for exploitation via optical imaging, the number of commmercially availible fluorophores for NIR imaging remain limited. In particular, simultaneous imaging of disease progression and functional imaging of more specific biological processes within the same sample is complicated by the requiste for multiple filtersets for fluorophores with similar spectral properties. Subsequent “bleeding” fluorescence through filtersets is unavoidable precluding ones ability to quantify specific fluorophores based on fluorescence. Similarly, descrimination of in vivo autofluorescence of similar spectral properties to commonly employed NIR dyes, consequent of ingested food comlicates contrast even further. More recently spectral imaging techniques have aided discrimination of fluorophores of similar spectral profiles however, these techniques attenuate much of the light reaching the detector. Time-domain (TD) optical imaging through the use of pulsed laser diodes and time resolved detector system, typically a photo-multiplier tube (PMT), has previously been demonstrated to distinguish between changes in physiological processes such as; tissue pH or calcuim concentration, based on changes in fluorescence lifetime of a fluorescently labelled probe. Here we demonstrate employing a single wave lenght TD optical imaging (eXplore Optix™, ART Inc) the potential to discriminate and quantify combinations of diverse NIR probes of spectrally similar properties but differing fluorescence lifetime on the basis of fluorescence lifetime in appropriate in vitro phantoms. Similarly, we illustrate the ability of this technique to discriminate between endogenous autofluorescence from administered fluorophores in vivo of leukemic cells in liver and spleen, and subsequent distinction of mixtures these fluorophores via their inherent fluorescent lifetimes in vivo.