High-resolution imaging in two-photon excitation microscopy using in situ estimations of the point spread function

We present a technique for improving the spatial resolution of two-photon excitation microscopy; our technique combines annular illumination with an in situ estimation of the point spread function (PSF) used for deconvolution. For the in situ estimation of the PSF, we developed a technique called autocorrelation scanning, in which a sample is imaged by the scanning of two excitation foci that are overlapped over various distances. The image series obtained with the variation of the distance between the two foci provides the autocorrelation function of the PSF, which can be used to estimate the PSF at specific positions within a sample. We proved the principle and the effectiveness of this technique through observations of a fluorescent biological sample, and we confirmed that the improvement in the spatial resolution was ~1.7 times that of typical two-photon excitation microscopy by observing a mouse brain phantom at a depth of 200 µm.