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Light scattering leads to a severe loss of axial and transverse resolution with depth into tissue, limiting accuracy and value of biomedical luminescence imaging techniques. High-resolution imaging beyond a few-millimeter depth is prohibited because diffusive transport dominates beyond a few scattering distances. In this study, light sheet imaging through scattering media is demonstrated using a radiotherapy linear accelerator to deliver well-defined thin scanned sheets of x-rays. These sheets produce Cherenkov light within the medium, which in turn excites luminescence of an optical probe across the sheet plane. This luminescence can then be imaged by an intensified camera positioned perpendicular to the sheet plane. The precise knowledge of the light sheet position within the medium allowed for efficient attenuation correction of the signal with depth as well as spatial deconvolution of the excitation light. Together these methods allowed for the first time, to the best of our knowledge, high-resolution imaging of tissue-equivalent phantoms up to 3 cm thick, yielding the precise position and shape of luminescent lesions located deep in tissue without the need for nonlinear image reconstruction.

Ionizing radiation is commonly delivered by medical linear accelerators (LINAC) in the form of shaped beams, and it is able to induce Cherenkov emission in tissue. In fluorescence-based microscopy excitation from scanned spots, lines, or sheets can be used for fast high-resolution imaging. Here we introduce Cherenkov-excited luminescence scanned imaging (CELSI) as a new imaging methodology utilizing 2-dimensional (∼5-mm-thick) sheets of LINAC radiation to produce Cherenkov photons, which in turn excite luminescence of probes distributed in biological tissues. Imaging experiments were performed by scanning these excitation sheets in three orthogonal directions while recording Cherenkov-excited luminescence. Tissue phantom studies have shown that single luminescent inclusions ∼1 mm in diameter can be imaged within 20-mm-thick tissue-like media with minimal loss of spatial resolution. Using a phosphorescent probe for oxygen, PtG4 with the CELSI methodology, an image of partial pressure of oxygen (pO2) was imaged in a rat lymph node, quantitatively restoring pO2 values in differently oxygenated tissues.