Study On Two-photon Fluorescent Viscosity Probes And Its Imaging In Living Cells

Since Denk developed the two-photon fluorescence three-dimensional imaging technique, numerous researches on two-photon bioimaging of live cells and tissues have been being carried out. Compared with conventional light microscope, fluorescence microscope and laser scanning confocal microscope, two-photon fluorescence microscope has many advantages such as higher spatial resolution, deeper penetration depth, lower tissue autofluorescence and less photodamage. Two-photon fluorescence microscope requires fluorescent probes have larger two-photon excited fluorescence action cross section (φ×δ), that is an important difference with common or confocal microscope. However, the relative laggard reaearch work on two-photon biofluorescence probes restricts the application of two-photon fluorescence microscope in the detection and imaging of living cells and tissues. Therefore, it is of great importance and significance to develop two-photn biofluorescence probes with large φ×δ.Now the development of two-photon biofluorescence probes has become an international concern.The value of microviscosity in different compartments of cells is different. The value of cytoplasm viscosity is1-2cp, very close to that of water, while the value of microviscosity near the intracellular membrane system can be up to140cp. Intracellular viscosity strongly influences intracellular transportation of mass and signal, interactions between biomacromolecules, and diffusion of reactive metabolites. For example, the changes of cytoplasm viscosity may affect the biological activity of the myocardial cells and lung macrophages, while the increasing of the viscosity of blood cell membrane will accelerate the aging. Therefore, detection of microviscosity at the cellular level is an important scientific proposition. Although there are many fluorescent viscosity probes have been reported, there is rare documents about fluorescent viscosity probes which could be applied to living cells. There is no report on two-photon fluorescence viscosity probe that could imaging in living cells. Because the distribution of the probe’s concentration within the cells is unkown and the fluorescent intensity is related to the concentration of the probe, Fluorescent viscosity probes, based on the methord only by detecting fluorescence intensity, could only give images of high-viscosity regions in the cells, but can not give the map of intracellular viscosity gradient distribution. However, the ratio fluorescence viscosity probes can not only eliminate probe load and data distortion caused by equipments, but also be able to give the gradient distribution of the local microviscosity.In our research work, we first find that probe Al is a two-photon fluorescence viscosity probe with red-emission. And then, we also speculate that probe A1is a single-photon ratio fluorescence viscosity probe, according to the experimental and theoretical analysis. Based on the analysis of two-photon fluorescent property of probe A1, we further come up with the idea that probe A1could to be a two-photon ratio of fluorescence viscosity probe.In addition, in view of physiological functions and the roles of homocysteine in vivo, for example, plasma homocysteine excess may induce many cardiovascular diseases, we also find that probe A7could specially identificate homocysteine in DMSO and that the interaction of probe A7with Hcy in cosolvent DMSO-PBS (v/v,8:2) could be observed by the naked eye.