Improving laser standards for three-photon microscopy

Abstract

. 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.

Tutorial: multiphoton microscopy to advance neuroscience research

“…initially broadening the pulses with the opposite sign of dispersion) such that the pulses subsequently compress as they propagate through the system to become transform-limited exactly at the focus point. It has been shown that for 3PE microscopy (typically requiring shorter excitation pulses but allows fordeeper imaging depths), even third-order-dispersion (TOD) is of concern [82], requiring pre-compensation using a pair of grism stretcher-compressors [83].…”

“…These lasers use low repetition rates (1 to 10 MHz) to increase the peak intensity and reduce the amount of heat delivered to the sample. Laser performance parameters including pulse shape, pulse energy, and pulse-to-pulse intensity variability used in 3PE microscopy should be occasionally measured to ensure optimal performance [82].…”

The influence of laser pulse shape and cleanliness on two-photon microscopy

“…which is known to cause pulse broadening 7 . This is evidently shown in Figure 2(d,e) revealing that the laser pulses cannot be efficiently compressed to their theoretical limits due to the phase distortions.…”

Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy

“…25 Pulse characterization with FROG allows for the recovery of spectral intensity and phase using iterative spectrogram inversion algorithms and provides a ground-truth pulse width measurement. 26,27 We optimized the prism pulse compressor at 1300 nm to achieve 51 fs pulses and a pulse duration of 51 to 75 fs for 1225 to 1360 nm, except for 1250 nm, which was 104 fs [Fig. 1(b)].…”