New two-photon fluorescent probe for multiphoton microscopy in biological media

An important ingredient in improving Multi Photon Laser Scanning Microscopy, MPLSM, is the development of efficient two-photon fluorescent (TPF) probes. We previously reported on a new class of TPF probes, specifically designed in order to maximize their efficiency in potential MPLSM applications. The fluorophores are based on a tetraketo derivative (TK) with a symmetric structure Donor-Acceptor-Donor (D-A-D). Those fluorophores have the following properties: a) Very large two-photon absorption coefficients (δ ∼ 1000GM); b) Two-photon excitation (TPE) peak wavelength strongly shifted to the red (λ ∼ 1μm); c) High fluorescence quantum efficiency; d) Large Stokes shifts of the fluorescence bands. We extended our work to a new fluorophore from this class that is more suitable for biological settings. This new fluorophore has a structure of crown-TK-crown that incorporates the ability to trap metal ions such as calcium. The TPE wavelength dependence of the TK-crown derivative is very similar to its analogous linear derivative with enhancement in the value of the cross-section, due to the stronger donor moieties. The TPE cross-section for the TK-crown derivative was about δ = 950 GM at λ_max = 980 nm.