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

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, and conventional hematoxylin-eosin (HE) histology.

The FM was constructed from an optimized concentration of commercially available human serum albumin, 700- and 800-nm NIR fluorophores, (99m)Tc-pertechnetate, dimethyl sulfoxide, and glutaraldehyde. Lymphangioleiomyomatosis cells coexpressing the sodium iodide symporter and green fluorescent protein were labeled with 700-nm fluorophore and (99m)Tc-pertechnatate and 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 costaining, autoradiography, and HE staining on the same slide using the FMs to coregister all datasets.

Optimized FMs, composed of 100 μM unlabeled human serum albumin, 1 μM NIR fluorescent human serum albumin, 15% dimethyl sulfoxide, and 3% glutaraldehyde in phosphate-buffered saline (pH 7.4), were prepared within 15 min, displayed homogeneity and stability, and were visible by all imaging modalities, including HE 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, autoradiography, and HE histopathologic staining.

When multimodal FMs are combined with a new technique for simultaneous same-slide NIR fluorescence imaging, autoradiography, and HE staining, macroscopic in vivo images can now be studied unambiguously at the microscopic level.