Physics

Biotechnology

Fluorescence microscope

Image resolution

In vivo

Laser

Biology

Optics

Resolution (logic)

Microscopy

Biomedical engineering

Femtosecond

Beam (structure)

Artificial intelligence

Materials science

Penetration depth

Computer science

Two-photon excitation microscopy

Fluorescence-lifetime imaging microscopy

Bessel beam

Preclinical imaging

Microscope

Medicine

Fluorescence

Biomedical Optics Express

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

Rapid volumetric imaging with Bessel-Beam three-photon microscopy