Three-photon fluorescence microscopy with an axially elongated Bessel focus

Area of Science:

• Neuroscience
• Biomedical Optics
• Microscopy

Background:

• High spatiotemporal resolution imaging is crucial for neuroscience research.
• Two-photon microscopy with Bessel beams offers advantages for volumetric imaging.
• Need for simpler, flexible, and robust volumetric imaging tools persists.

Purpose of the Study:

• To demonstrate three-photon fluorescence imaging utilizing an axially extended Bessel focus.
• To adapt Bessel beam generation for advanced volumetric imaging applications.
• To evaluate the utility of this technique for neural imaging in biological samples.

Main Methods:

• Implementation of an axicon-based module to generate Bessel foci.
• Modification of two-photon fluorescence microscopy for three-photon excitation.
• Application of the extended Bessel focus for imaging neural structures.

Main Results:

• Successful demonstration of three-photon fluorescence imaging with an axially extended Bessel focus.
• Generation of Bessel foci with tunable numerical apertures and axial lengths.
• Effective application of the technique for imaging mouse brain slices and in vivo mouse brains.

Conclusions:

• Three-photon fluorescence imaging with an extended Bessel focus is a viable volumetric imaging tool.
• The axicon-based system provides flexibility in generating Bessel foci for microscopy.
• This method advances the capability for high-resolution neural network imaging in neuroscience.