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

Abstract

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.

A conceptual review on Bessel beams

“…The schematic diagram of BB generation with axicon under the HoG beam pump is shown in figure 24. The HoG beam is a doughnut-shaped beam with no helical wave-front [121] and it can be easily synthesized with SLM or SPP [122][123][124][125][126]. As shown in the below figure 24, first we can pass the Gaussian beam through the SPP of topological charge l to produce a Gaussian vortex beam of order l and further this beam can pass through the second SPP of topological charge, -l. The second SPP creates azimuthal phase retardation in the opposite direction to the first SPP.…”

“…Both the diffractive optical elements provide the maximum conversion efficiency. The experimentally generated optical field amplitude of the HoG beam of order m is in the form of [122,127]…”

Polarized Shack-Hartmann wavefront sensor

“…Many applications show that the SHWFS can meet a high accuracy and achieve a stable property under the night or the other low background or stray light scene. However, under the bright background light scenes such as the above conditions of daytime stray light and corneal reflex, the signal-to-background ratio (SBR) [9] or signal-to-noise ratio (SNR) of each sub-aperture of SHWFS will be limited obviously, where SBR usually used to indicate the intensity level of the background light. Under ultra-worse condition, one may not find any signal light from the SHWFS, which limit its measurement ability of wavefront aberration.…”