Abstract
Improving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to find the best balance between signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging.
Highlights
Multiphoton microscopy has demonstrated unique advantages for deep and live tissue imaging [1]
If photodamage had been sensitive to the fluorophore, we would have obtained significantly higher values of PNL and n in the case of unlabeled embryos. These results demonstrate that increasing the signal in 2P-SPIM by increasing the average power or decreasing the laser is limited by irreversible highly nonlinear photodamage with an order close to n = 5, which are not mediated by the fluorophore
We achieved high-speed multiphoton imaging in vivo while maintaining both low laser average power and peak intensity
Summary
Multiphoton microscopy has demonstrated unique advantages for deep and live tissue imaging [1]. The acquisition speed of standard point-scanning two-photonexcited fluorescence (2PEF) microscopy is generally bounded to a μs per pixel due to signal limitations resulting from fluorophore photophysics [2] This limit sets important constraints for investigating fast biological phenomena or for multiscale imaging [3]. Among the strategies developed to improve the acquisition speed in multiphoton fluorescence imaging, light-sheet illumination exhibits distinctive advantages [4], with applications in cell biology [10], neurosciences [11], developmental biology [12], or organoid research [13]. The parallelization of the illumination results in long pixel dwell times and in higher signal levels compared to fast scanning approaches [8] These characteristics are expected to set different photoperturbation-related constraints in multiphoton light-sheet microscopy compared to fast collinear geometry approaches
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