Biodistribution Monitoring of Fluorescent Markers In Vivo Using the iBox Explorer2 Imaging Microscope

Biodistribution studies are critical in the investigation of novel pharmacological agents, yielding vital information about tissue distribution of drug formulations as well as to characterize non-pharmaceutical agents, particularly in an in vivo model. Fluorescent markers, either in the form of genetic reporters like green fluorescent protein (GFP), NIR dyes, organic/inorganic dyes or nanoparticles, can be attached to the molecule of interest. UVP LLC's iBox® Explorer™2 Imaging Microscope is a versatile instrument capable of monitoring the distribution of fluorescent materials throughout organs and tissues of small animals in vivo. The iBox Explorer2 is well suited for imaging the distribution of dye in a whole animal and allows:

• Non-invasive monitoring of fluorescence in vivo
• Rapid detection of low fluorescent signals with a high sensitivity CCD camera
• Anesthesia and warming of experimental animals for safe and rapid imaging
• Macro to micro detection from the whole animal to the cellular level

To highlight the capabilities of whole animal imaging, a biodistribution study was conducted using a near infrared (NIR) dye conjugated to an antibody. A dose of the conjugate was administered in an anesthetized animal and distribution of the dye monitored via fluorescence imaging over a 4-hour period.

Qualitative and quantitative data analysis revealed the NIR dye conjugated to anti-CEA monoclonal antibody (MAb) is seen concentrating in the liver of the mouse (Figure 1) over a 4 hour period. At time 0 there was no detectable NIR signal. At 10 minutes post-injection, dye rapidly accumulated in the liver and the vasculature running along the vertebral column. Post-experiment surgical exposure (Figure 2: highlighting liver [top], bladder [bottom] and region of local inflammation [left]) revealed presence of dye in the liver, bladder and region of inflammation in subcutaneous tissue. In addition, ex vivo analysis of vital organs confirmed that much of the dye had been sequestered by the liver.

The observed rapid dye accumulation in both the liver and lymph node shows a linear increase in intensity over time, suggesting sequestration of dye in this organ in order to clear the conjugate from the systemic circulation. It is also possible that accumulation of the dye is a result of its unique chemical structure. Furthermore, presence of dye in the neck lymph nodes suggests expression of surface antigen CEA or non-specific binding of this antibody to the reticular cells. Taken together, these data show possible mechanisms for uptake of dye in this nude mouse model and can form the foundation for future biodistribution studies.

The iBox Explorer2 is an ideal system for imaging distribution of fluorescent markers within small animals for pre-clinical experimental protocols. The wide-angle lens allows for the imaging of a whole animal and can accommodate up to two mice. In addition, the cooled, scientific-grade CCD camera rapidly captures crisp, publication quality images. A bright xenon arc lamp provides intense excitation light from the visible to NIR range, accommodating a host of in vivo studies and fluorescent markers. An application-specific filter set can be tailored for imaging virtually any pre-clinical fluorescence study.

For the full application note and video about UVP's iBox Explorer, click here.