Significance: Three-photon excitation microscopy has double-to-triple the penetration depth in biological tissue over two-photon imaging and thus has the potential to revolutionize the visualization of biological processes in vivo. However, unlike the plug-and-play operation and performance of lasers used in two-photon imaging, three-photon microscopy presents new technological challenges that require a closer look at the fidelity of laser pulses.

Aim: We implemented state-of-the-art pulse measurements and developed innovative techniques for examining the performance of lasers used in three-photon microscopy. We then demonstrated how these techniques can be used to provide precise measurements of pulse shape, pulse energy, and pulse-to-pulse intensity variability, all of which ultimately impact imaging.

Approach: We built inexpensive tools, e.g., a second harmonic generation frequency-resolved optical gating (SHG-FROG) device and a deep-memory diode imaging (DMDI) apparatus to examine laser pulse fidelity.

Results: First, SHG-FROG revealed very large third-order dispersion (TOD). This extent of phase distortion prevents the efficient temporal compression of laser pulses to their theoretical limit. Furthermore, TOD cannot be quantified when using a conventional method of obtaining the laser pulse duration, e.g., when using an autocorrelator. Finally, DMDI showed the effectiveness of detecting pulse-to-pulse intensity fluctuations on timescales relevant to three-photon imaging, which were otherwise not captured using conventional instruments and statistics.

Conclusions: The distortion of individual laser pulses caused by TOD poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation. Moreover, an acceptably low pulse-to-pulse amplitude variability should not be assumed. Particularly for low repetition rate laser sources used in three-photon microscopy, pulse-to-pulse variability also degrades image quality. If three-photon imaging is to become mainstream, our diagnostics may be used by laser manufacturers to improve system design and by end-users to validate the performance of their current and future imaging systems.