Background: Fluorescence molecular imaging is a valuable imaging modality at both the cellular and the whole-body imaging regimes. Although valuable for pathological analysis, fluorescence histology has a severely limited field of view. Similarly, bulk fluorescence imaging (such as with epi-illumination schemes) is either highly surface weighted, or limited to a resolution of a few cubic millimeters at best using tomographic approaches. Cryofluorescence tomography (CFT) is proposed as an imaging scheme to bridge the gap between microscopic and mesoscopic imaging methods, while still exploiting the vast and growing library of fluorescent tracers.
CFT is comprised of serial cryoslicing and high resolution imaging of a tissue sample block at multiple visible and NIR wavelengths. For each slice, a series of fluorescence images is captured simultaneous with white light images. In this way, valuable molecular fluorescence information is gathered along with anatomical information. Furthermore, imaging is performed on the block face rather than on transferred slices, thus completely eliminating registration artifacts caused by tissue deformation, folding, and tearing. Imaging is performed by interfacing a cryoslicer (Leica 3050s) with a surgical fluorescence imaging system (FLARE model R1, Curadel, Marlborough, MA). This imaging scheme can be used to automatically recover 3D fluorescence molecular information at mesoscopic resolution, with imaging times of under an hour for a whole mouse.
Materials and Methods: To test this imaging scheme, a rat was injected intrathecally with a near-infrared fluorophore having peak emission at 800 nm and intravenously with a near-infrared fluorophore having peak emission at 700 nm. At one hour, the kidneys were resected and imaged using CFT with 25 micron slices to be able to follow tracer clearance from the CSF to the blood and blood to urine using near-infrared fluorescence. Basic structural analysis based on the white light images was also performed. A color-based segmentation was performed to segment the vasculature of the sample.
Results: A comparison of the different imaging channels (Figure) shows a snapshot comparison of the renal clearance of the two tracers through different injection routes at approximately 25 micron voxel size. The images show that it is possible to resolve molecular and structural features on the same order of magnitude as lymphatic channels and the vasculature.
Conclusions: Using this new CFT system, two independent biomarkers can be imaged simultaneously over an entire mouse or rat with resolution far superior to whole-body nuclear imaging methods. By utilizing FLARE® lipophilic cell tracking dyes, single cells can be labeled and tracked anywhere in the animal. This should be particularly useful for tracking the migration and homing of metastatic cells. Using NIR fluorescent targeting ligands, virtually any in vivo target can be assessed over large volumes of tissue. Multiple examples of CFT for imaging cancer metastasis, lymphovascular processes, and biomarkers will be presented.