Whole-body fluorescence cryotomography identifies a fast-acting, high-contrast, durable contrast agent for fluorescence-guided surgery.

Imaging of tumor-specific fluorescent contrast agents to guide tumor removal has been shown to improve outcomes and is now standard practice for some neurosurgical procedures. However, many agents require administration hours before surgery, a practical challenge, and may exhibit inconsistent concordance with contrast-enhanced MRI (CE-MRI), the current standard for diagnosing and guiding glioma removal. A fluorescent agent that accurately marks tumor shortly after administration and is otherwise similar to CE-MRI would help overcome these shortcomings. We used whole-body 3-D fluorescence cryo-imaging and co-registered CE-MRI volumes to evaluate several fluorescent contrast agent candidates for diagnostic performance and concordance with CE-MRI. Mice with brain tumors were administered a cocktail of fluorescent agent candidates and a MRI contrast agent, and then imaged with MRI and fluorescence cryo-imaging at several timepoints after administration. The high-resolution 3-D cryo-imaging volumes of the fluorescent agents were used to determine diagnostic performance metrics and correlation with CE-MRI. While all agents showed positive metrics, one agent, tetramethylrhodamine conjugated to a small polyethylene glycol chain (TMR-PEG1k), outperformed the others, exhibiting minimal normal brain signal, high tumor-to-background-ratio, diagnostic accuracy, and cross-correlation to CE-MRI at all post-administration timepoints (10-90 min) and tumor lines examined. These favorable properties establish TMR-PEG1k as a promising candidate for surgical guidance.