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.
Highlights
The ubiquitous field of two-photon microscopy has been used to visualize a wide array of biological phenomena in vivo, from imaging neurons in healthy animals[1,2,3] and in disease models such as Alzheimer’s,4 to imaging the movement of fluorescently labeled viruses invading the lungs[5,6] and the mechanisms of glomerular filtration in the kidneys.[7]
The distortion of individual laser pulses caused by third-order dispersion (TOD) poses significant challenges to three-photon imaging by preventing effective compression of laser pulses and decreasing the efficiency of nonlinear excitation
For low repetition rate laser sources used in threephoton microscopy, pulse-to-pulse variability degrades image quality
Summary
The ubiquitous field of two-photon microscopy has been used to visualize a wide array of biological phenomena in vivo, from imaging neurons in healthy animals[1,2,3] and in disease models such as Alzheimer’s,4 to imaging the movement of fluorescently labeled viruses invading the lungs[5,6] and the mechanisms of glomerular filtration in the kidneys.[7]. Since three-photon excitation requires the near-simultaneous absorption of three photons, laser pulses with higher photon density (higher energy and shorter pulse duration) are used when compared to two-photon microscopy. As a result of using higher energy pulses, lower repetition rates must be used to manage the amount of heat delivered to the sample. The integrated fluorescence signal used to construct imaging pixels is a result of fewer excitation pulses. Pulse-to-pulse intensity fluctuations on the timescale of microseconds to milliseconds to seconds will degrade the imaging quality of biological samples. Because the three-photon absorption rate is proportional to intensity cubed,[13] distortion of individual pulses in the excitation source can detract from excitation efficiency. Examining time-dependent biological processes that produce meaningful changes in fluorescence brightness requires a stable, distortion-free three-photon excitation source
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