Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes

The fluorescence imaging of tissue is essential for studying biological events beyond the cellular level. Twophoton microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resolution imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomolecules. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examined, the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is observed not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-soluble, and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the bluegreen, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.