MPE IMAGING OF DRUG LOCALIZATION AND METABOLISM:

Tissue autofluorescence images of a perfused mouse colon: MPE Imaging of Drug Localization & Metabolism

5-aminosalicylic acid (5-ASA) localization and metabolism in the colon. 5-ASA has been the anti-inflammatory agent of choice for ulcerative colitis for over 50 years, yet little is about its mode of action. In collaboration with M.H. Montrose at Johns Hopkins Medical School, we applied nonlinear microscopy to this problem. Using MPE-LSM the uptake of 5-ASA and the rate of conversion to acetyl-5- ASA (metabolized 5-ASA) can be measured in living colon tissue samples in a two-layer perfusion chamber (see below). The therapeutic concentration of ASA is 30mM. Due to absorption events throughout the beam with conventional illumination, confocal microscopy only allows penetration to ~20 µm. MPE occurs only in the focal volume; the 5-ASA is transparent to the 700 nm excitation.

The rate of metabolism of 5-ASA to Ac-5-ASA in the crypts can be measured ratiometrically by using the emission shift of the acetylated form (see spectra at left). The image below shows the fluorescence ratio (F440/F505) change that occurs after the addition of 30 mM 5-ASA to the perfusion solution at 0 minutes. With 30 mM 5-ASA in the perfusion bath this measurement was not possible using a single photon UV confocal due to the severe attenuation of the excitation beam.