Enzyme Activity Imaging Using Activatable Raman Probes

Raman imaging, which detects molecular vibration, has attracted significant attention recently as an imaging technique with superior capability for multiplexed detection compared to fluorescence imaging. Raman imaging was originally proposed as a label-free method, but the recent development of vibrational tags such as small alkynes or Raman probes for multiplexed imaging enabled us to visualize a variety of biomolecules with high specificity and improved sensitivity, thus the biocompatibility of Raman imaging has been dramatically improved. We have been working on developing an activatable Raman imaging probe whose Raman signal is activated upon reaction with target enzymes. We utilized the specific phenomenon of the signal intensity of stimulated Raman scattering (SRS) increasing remarkably under electronic pre-resonance (EPR) conditions, in which a molecule is excited at a wavelength of 100-200 nm longer than its molecular absorption. Firstly, we focused on 9CN-JCP, one of the pyronin derivatives with nitrile at the 9th position (9CN-pyronin) as a scaffold dye, and developed multicolor activatable Raman imaging probes that can detect plural enzyme activities simultaneously in living cells. More recently, we focused on rhodol derivatives with nitrile at the 9th position (9CN-rhodol), which tend to form aggregates in aqueous solution than 9CN-pyronins, and we developed novel activatable Raman probes for enzymes that can form aggregates upon reaction with target enzymes, enabling us to perform ex vivo imaging of enzyme-expressing cells or regions in live Drosophila tissues. In this review, we first give an overview of recent trends in biological Raman imaging, and then introduce our research achievement on the development of activatable Raman probes.