People have been interested to observe the inside of intact cells or tissues for a long time. However, it is challenging to get the high spatial-resolution deep tissue image due to the strong scattering of most biological samples in conventional microscopy. Multi-photon fluorescence microscopy is a useful tool for whole cell and in-vivo tissue imaging, because it overcome this problem with near-infrared light region that provides less scattering, less phototoxicity and deep penetration depth. Moreover, intrinsic small excitation volume generation of multi-photon excitation process enables optical sectioning for 3D image reconstruction.
In this poster, we report the recent progress in three-photon-induced fluorescence (3PIF) microscopy. Ultimately, we would like to report the 3D imaging by the 3PIF from tryptophan (Trp), a potential intrinsic fluorescent dye in a biological system. Trp has absorption and emission peaks at 280 nm and 350 nm, respectively. These UV absorption and emission have hindered people from using Trp as fluorescent dye. We expect to avoid this problem by three-photon absorption process.
For showing our microscopy 3PIF PSF, we used 100 nm fluorescent bead that has UV absorption and visible emission peak at 260 nm and 450 nm, respectively. It shows that our microscopy has xxx nm lateral resolution and xxx nm axial resolution at 1000 nm excitation wavelength through 60x objective lens (water immersion, NA 1.2). Also to show the 3D imaging ability, we imaged glass beads in Trp monomer solution .