Single pixel hyperspectral Cherenkov-excited fluorescence imaging with LINAC X-ray sheet scanning and spectral unmixing.

Cherenkov light induced from megavolt (MV) X-rays during external beam radiotherapy serves as an internal light source to excite phosphors or fluorophores within biological tissues for molecular imaging. The broad spectrum of Cherenkov light leads to significant spectral overlap with any luminescence emission and, to overcome this problem, a single pixel hyperspectral imaging methodology was demonstrated here by coupling the detection with light sheet scanning and filtered back projection reconstruction of hyperspectral images. Thin scanned sheets of MV X-rays produce Cherenkov light to illuminate the planes deep within the tissue-simulating media. A fluorescence probe was excited by Cherenkov light, and a complete hyperspectral sinogram of the data was obtained through translation and rotation of the beam. Hyperspectral 2D images finally were reconstructed. Through this approach of spectral unmixing, it was possible to resolve hyperspectral images of both the Cherenkov and resulting fluorescence intensity from molecular sensors.