Time-resolved nanoscale imaging of biomolecules in living cells and tissues: Prospects for small animal imaging

A major need in non-invasive optical imaging of small animal models is an ability not only to visualize the solid tumors in vivo but to reproducibly quantify the tumor burden and its propensity to metastasize to other organs of the body. It is crucial to non-invasively detect the subtle molecular changes that can make a cell 'abnormal' and cancerous in its very early stage. Currently available methods for non-invasive optical imaging of solid tumors in small animals employ intensity-based detection that are severely affected by spectral artifacts and ubiquitous autofluorescence background. Thus these approaches serve merely as visualization tools and are unable to precisely quantify the size and shape of the tumors in vivo. There is a growing need to establish a reliable, reproducible and non-invasive optical imaging methodology that can provide quantitative information on solid tumors in vivo. This manuscript addresses this vital issue and proposes to employ fluorescence lifetime (rather than intensity) as a contrast parameter to discriminate tumor tissue expressing green fluorescent protein (EGFP) from surrounding autofluorescence background. In this manuscript, we present accurate lifetime measurements in intact living cells and ex vivo tissues and propose that this methodology is a potentially vital approach for whole small animal imaging.