Point-Spread-Function and Deep-Tissue Imaging
in Two-Photon Fluorescence Microscopy
Chen-Yuan Dong1, Karsten Koenig2, and Peter T. C. So3
1Department of Physics, National Taiwan University, Taipei 106, Taiwan, R.O.C.
2Friederich-Schiller University Jena, Institute of Anatomy II, Teichgraben 7, 07743 Jena, Germany
3Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
In recent years, two-photon fluorescence microscopy has become a major branch of modern optical imaging technology. Based on non-linear excitation of fluorescent molecules, two-photon microscopy allows confocal-like sectioning in acquired images and improved depth penetration. Specifically, the near-infrared wavelengths used for sample excitation are absorbed and scattered less by tissue than the corresponding one-photon wavelengths. As a result, the excitation photons can be delivered to the specimen more deeply. In this work, we examined the two-photon image resolution in turbid medium by a direct measurement of the point-spread-function (PSF). We found that up to an imaging depth of 200 mm, there is no evidence of PSF degradation in tissue-like, scattering specimen. We also compared the performance of water and oil immersion objectives in excised human skin, an optically complex sample. And we found that both objectives performed similarly in the skin specimen. These studies are important for developing two-photon fluorescence microscopy into an effective, quantitative bioimaging technique.