Two-photon microscopy with enhanced resolution and signal-to-background ratio using hollow Gaussian beam excitation.

Two-photon microscopy (TPM) offers deeper imaging depth inside the scattering medium, however, it suffers from limited resolution owing to the longer excitation wavelength. We demonstrate the use of a hollow Gaussian beam (HGB) at the therapeutic window to improve the resolution and signal-to-background ratio (SBR). The HGB was produced by omitting the azimuthal phase term from the vortex mode, and the excitation point spread function (PSF) can be readily tuned by the mode order. The performance of the TPM with HGB was evaluated by experimentally imaging 100 nm fluorescent beads to estimate the PSF. The HGB improved the lateral resolution of the TPM by 36% in contrast to the conventional TPM. The HGB also furnishes an improvement of SBR by eliminating the out-of-focus light owing to its ring shape. Furthermore, we have used a translating lens-based module for additional lateral resolution tuning and reduced the resolution further down to 44% with respect to conventional TPM. Finally, we have performed imaging with merely two-dimensional scanning of a 50 µm thick mouse brain slice (Thy-YFP H-line) using the developed TPM with HGB. Our compact, robust, and low-cost design of the HGB generation scheme can easily be integrated into the commercial TPM to accommodate the improvements.