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

[lymphangioleiomyomatosis]

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 .T he FM was constructed from an optimized concentration of commercially available human serum albumin , 700 - and 800 -nm NIR fluorophores , (99 m )Tc-pertechnetate , dimethyl sulfoxide , and glutaraldehyde . Lymphangioleiomyomatosis cells coexpressing the sodium iodide symporter and green fluorescent protein were labeled with 700 -nm fluorophore and (99 m )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 .W hen 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 .