University of Tokyo researchers have developed a novel imaging technique which allows for high-resolution 3D enzyme imaging in whole, cleared organs. The technique is thanks to new probe molecules which can anchor fluorescent dyes as they are activated by enzymes.
Traditional imaging of enzyme activity is conducted using fluorescence probes; however, whole organs cannot be 3D mapped as fluorescent light barely penetrates tissue. To remedy this a technique called clearing can be employed which makes tissues transparent while maintaining their structure using a mix of different solvents and reagents. However, during the intensive washing needed for clearing to work small molecules like fluorescent probes are typically washed out of the tissue.
The innovative technique published in Angewandte Chemie International Edition, remedies this issue by employing a specially developed probe molecule which consists of a dye, and anchoring unit, and an amino acid group.
To prove the method, the team focused on aminopeptidase N (APN), an enzyme involved in various physiological processes and tumor development. Using their method, in regions containing active APN, the enzyme will split the amino acid off the probe to activate the anchoring unit. This will solidly anchor the fluorescent probe to the surrounding tissue so that it is not washed away.
Using this method, the team obtained high resolution 3D maps of APN activity using fluorescence microscopy. In addition to the maps, the team also visualized the difference in APN activity within tubular structures of the kidney.
By obtaining 3D high resolution maps of enzyme activity, the method developed opens the door for unbiased drug development evaluation which does not overlook activity which occurs at the cellular level in whole organs.