Microscopic validation of macroscopic in vivo images enabled by same-slide optical and nuclear fusion

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It is currently difficult to determine the molecular and cellular basis for radioscintigraphic signals obtained during macroscopic in vivo imaging. The field is in need of technology that helps bridge the macroscopic and microscopic regimes. To solve this problem, we developed a fiducial marker (FM) simultaneously compatible with 2-color near-infrared (NIR) fluorescence (700 and 800 nm), autoradiography (ARG), as well as conventional hematoxylin and eosin (H&E) histology. Methods: The FM was constructed from an optimized concentration of commercially available human serum albumin (HSA), 700 nm and 800 nm NIR fluorophores, 99mTc-pertechnatete, DMSO, and glutaraldehyde (GA). Lymphangioleiomyomatosis (LAM) cells co-expressing the sodium iodide symporter (NIS) and green fluorescent protein (GFP) were labeled with 700 nm fluorophore and 99mTc-pertechnatete, then administered intratracheally into CD-1 mice. After in vivo SPECT imaging, and ex vivo SPECT and NIR fluorescence imaging of the lungs, 30 μm frozen sections were prepared and processed for 800 nm NIR fluorophore co-staining, ARG, and H&E staining on the same slide using the FMs to co-register all data sets. Results: Optimized FMs, composed of 100 μM unlabeled HSA, 1 μM NIR fluorescent HSA, 15% DMSO, and 3% GA in PBS (pH 7.4) were prepared within 15 min, displayed homogeneity and stability, and were visible by all imaging modalities, including H&E staining. Using these FMs, tissue displaying high signal by SPECT could be dissected and analyzed on the same slide and at the microscopic level for 700 nm NIR fluorescence, 800 nm NIR fluorescence, ARG, and H&E histopathological staining. Conclusion: When multimodal FMs are combined with a new technique for simultaneous same-slide NIR fluorescence imaging, ARG, and H&E staining, macroscopic in vivo images can now be studied unambiguously at the microscopic level.

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