Saturated two-photon excitation fluorescence microscopy with core-ring illumination

Area of Science:

• Microscopy and Imaging Technologies
• Biophotonics
• Optical Engineering

Background:

• Two-photon excitation microscopy (TPM) is crucial for deep tissue imaging.
• Achieving high spatial resolution in 3D TPM remains a challenge.
• Conventional TPM can suffer from out-of-focus excitation and sidelobe artifacts.

Purpose of the Study:

• To enhance the spatial resolution of two-photon excitation microscopy.
• To suppress sidelobe artifacts in the point spread function.
• To demonstrate improved 3D imaging of biological samples.

Main Methods:

• Combining saturated excitation (SAX) of fluorescence with pupil manipulation.
• Theoretical estimation of resolution improvement and sidelobe effects.
• Experimental validation using a tissue phantom at depths up to 100 μm.

Main Results:

• The combination of SAX and core-ring illumination effectively enhances 3D spatial resolution.
• Sidelobe artifacts in the point spread function were significantly suppressed.
• Experimental results demonstrated superior resolution compared to conventional TPM.

Conclusions:

• The proposed technique offers a significant advancement for high-resolution 3D imaging in biological tissues.
• This method is effective for deep tissue observation (up to 100 μm).
• The integration of SAX and pupil engineering provides a powerful tool for advanced microscopy applications.