Interleaving SLIDE microscopy

Abstract

Two-Photon microscopy (TPM) is an intravital imaging technique with deep tissue penetration, high three-dimensional resolution and low photobleaching. However, imaging speeds have traditionally been limited because images are generated by point scanning. Recently, Spectro-Temporal Laser Imaging by Diffractive Excitation (SLIDE) introduced a high-speed laser-scanning approach for two-photon microscopy, harnessing a swept-source laser in combination with a diffraction grating for inertia-free point scanning with line-rates at Megahertz (MHz) frequencies and frame-rates in the kilohertz range. Notably, the pixel-rate reached up to Gigapixels/s, which scales with ultra-short second harmonic generation signals. However, this nanosecond pixel dwell time is too short for fluorescence decays. Here, we present an adaptive approach to high-speed multiphoton imaging where the pixel-rate of the SLIDE microscope can be adapted to the fluorescence lifetime of the sample. This is achieved by interleaved sampling, allowing for larger temporal intervals in the time domain while preserving spatial sampling density. We demonstrate adaptive frame-rate SLIDE microscopy by flexible, active pulse modulation on an electro-optic modulator (EOM) and discuss how this can be extended to future smart SLIDE systems where the pixel dwell time can be optimally set for each sample.

The authors' abstract, as published at the source. Frontiers in Photonics, 2026 · DOI ↗