In Vivo Fluorescence Imaging Of Internally Illuminated Molecular Probes

Omics era has yielded a wealth of information on various macromolecules and signaling pathways that regulate various diseases. Elucidating their role in vivo is essential for stringent screening of the diverse players and pathways. Optical imaging, especially fluorescence imaging has emerged as a keen front runner in such discoveries of target molecules. Researchers utilizing the fluorescent molecular tools earlier used in the in vitro experiments, microscopy and cell culture, face some challenges attempting in vivo molecular imaging. Especially with molecular probes that fluoresce in the visible wavelengths. The key challenges include the attenuation of light by tissues, and light absorption by the endogenous macromolecules. We present here a methodology whereby the molecular signals from the visible range emitters in vivo are efficiently detected limiting skin autofluorescence. As a proof of principle, focused internal illumination was used to excite calcein with an endoscope introduced rectally. The emitted light was then detected externally using a camera. A 50-fold and higher increase in signal to noise ratio was observed, and with negligible interference by skin autofluorescence. We report here a novel near non-invasive molecular imaging approach capable of imaging molecular probes and fluorescence reporting tools that emit in the visible wavelengths with reduced tissue interference.