Two-photon scanned light sheet fluorescence microscopy with axicon imaging for fast volumetric imaging.

Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition.

Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM.

Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM.

Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz.

Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.