Enhancing the depth of tissue microscope imaging using two-photon excitation of the second singlet state of fluorescent agents

Increasing the depth to image inside tissue is critical in biomedicine. Two-photon (2P) excitation of the second singlet (S-2) state of a group of fluorescent agents with near infrared emission, Chlorophyll a (Chl a) and Indocyanine green (ICG), is used to extend the optical imaging regime of 2PM into "tissue optical window" for deep tissue penetration. The fast nonradiative from S-2 to S-1 yields both emission and absorption wavelengths in the therapeutic window. The salient feature is to place both the 2P excitation and emission wavelengths of the imaging agents falling into the "tissue optical window". As a first step to achieve deeper optical imaging, Chl a and ICG are investigated and demonstrated as imaging agents for 2P S-2 excitation microscope image.