Enhancing the axial resolution of two-photon imaging

An axial-resolution-enhanced two-photon laser scanning microscopy system is presented in this paper. In the proposed method, we use a spatial light modulator (SLM) for the phase modulation of the excitation light. The axially split point spread function (PSF) is generated by loading a 0-π pattern on the SLM. The final quality-enhanced images are acquired by subtracting the two consecutive images acquired by the original PSF and the split PSF. Because of the fluorescence differential processing, the axial elongation of the particles images is suppressed, and the axial resolution is enhanced accordingly. With the axial resolution enhanced, the overlap between layer images is also reduced, which decreases the background noise of the images and enhances the contrast and image quality of the acquired fluorescence images. The capability of axial resolution and contrast enhancement is successfully demonstrated by both theoretical calculation and experimental results.