Scanningless depth-resolved microscopy

TLDR

Laser‑scanning microscopy enables optical sectioning but suffers from reduced frame rates due to its serial acquisition process. The authors aim to show that spatiotemporal pulse shaping in multiphoton microscopy can provide full‑frame depth‑resolved imaging without scanning. Their approach relies on temporal focusing, compressing the pulsed excitation field at the focal plane and stretching it beyond. When applied to Drosophila egg chambers, the technique yields depth‑resolved two‑photon excitation fluorescence images with nearly 10⁵ effective pixels using a standard Ti:Sapphire laser oscillator.

Abstract

The ability to perform optical sectioning is one of the great advantages of laser-scanning microscopy. This introduces, however, a number of difficulties due to the scanning process, such as lower frame rates due to the serial acquisition process. Here we show that by introducing spatiotemporal pulse shaping techniques to multiphoton microscopy it is possible to obtain full-frame depth resolved imaging completely without scanning. Our method relies on temporal focusing of the illumination pulse. The pulsed excitation field is compressed as it propagates through the sample, reaching its shortest duration at the focal plane, before stretching again beyond it. This method is applied to obtain depth-resolved twophoton excitation fluorescence (TPEF) images of drosophila egg-chambers with nearly 105 effective pixels using a standard Ti:Sapphire laser oscillator.