Sensitivity and accuracy of hybrid fluorescence-mediated tomography in deep tissue regions

Fluorescence-mediated tomography (FMT) enables noninvasive assessment of the three-dimensional distribution of near-infrared fluorescence in mice. The combination with micro-computed tomography (µCT) provides anatomical data, enabling improved fluorescence reconstruction and image analysis. The aim of our study was to assess sensitivity and accuracy of µCT-FMT under realistic in vivo conditions in deeply-seated regions. Accordingly, we acquired fluorescence reflectance images (FRI) and µCT-FMT scans of mice which were prepared with rectal insertions with different amounts of fluorescent dye. Default and high-sensitivity scans were acquired and background signal was analyzed for three FMT channels (670 nm, 745 nm, and 790 nm). Analysis was performed for the original and an improved FMT reconstruction using the µCT data. While FRI and the original FMT reconstruction could detect 100 pmol, the improved FMT reconstruction could detect 10 pmol and significantly improved signal localization. By using a finer sampling grid and increasing the exposure time, the sensitivity could be further improved to detect 0.5 pmol. Background signal was highest in the 670 nm channel and most prominent in the gastro-intestinal tract and in organs with high relative amounts of blood. In conclusion, we show that µCT-FMT allows sensitive and accurate assessment of fluorescence in deep tissue regions.