Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

TL;DR

This paper presents a scanning-free, widefield two-photon fluorescence imaging technique enabling high-speed, low-photo-bleaching live cell microscopy suitable for observing rapid calcium dynamics in neurons.

Contribution

The authors demonstrate a widefield two-photon excitation method with increased field size and 100 Hz frame rate, reducing photo-bleaching compared to single-photon methods.

Findings

Achieved up to 100 Hz frame rate imaging

Observed significantly reduced photo-bleaching

Enabled high time-resolution live cell imaging

Abstract

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca$^{2+}$ events in primary rat neurone cultures loaded with the fluorescent Ca$^{2+}$ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.