Multicolored two-photon fluorescent microscopy and localized two-photon fluorescent spectroscopy in living cells

Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one-photon systems. Our lnstitute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two-photon laser scanning microscopy (TPLSM) to understand the real rime cellular transport of the chemotherapeutic agent, Lutenizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the intergration of two-photon and nanosized technologies. Nanoclinics (20-30 nm silica bubbles) can be fabricated to contain these two-photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.