KEYWORDS: Brain, Deep tissue imaging, Neuroscience, Neuroimaging, In vivo imaging, High speed imaging, High power lasers, Femtosecond pulse shaping, Femtosecond phenomena, Brain imaging
In this work, we customized a multi-photon spinning disc unit with a tunable high-power femtosecond laser to demonstrate the first three-photon spinning disk microscopy, achieving ~100Hz frame rate. We demonstrate its pioneering applications on green fluorescent beads, in vivo Drosophila brains, and fixed mouse brains. Furthermore, our findings from brain tissue imaging reveal that three-photon spinning disk imaging exhibits a significantly lower attenuation rate when compared to its two-photon spinning disk counterpart, paving the way of utilizing three-photon excitation on high-speed deep tissue imaging.
KEYWORDS: Signal to noise ratio, Deep learning, Two photon imaging, Image enhancement, Imaging systems, Education and training, Two photon excitation microscopy, Signal processing, Neurons, Neural networks
We developed a high-speed two-photon volumetric imaging system with hundreds of axial layers that match a deep-learning denoising model to capture millisecond-level functional changes in individual neurons with high SNR. Compare with general deep-learning methods, the spatial information-based training method not only enhances SNR by 300% but prevents temporal distortion. Our proof-of-concept experiment focused on calcium dynamics in cerebellum Purkinje cells, revealing similar responses in the parallel dendritic layers, yet significant divergence in the somatic area. This sheds light on the intricate signal processing at individual neuron levels, validating our imaging system.
We have demonstrated a widely-tunable femtosecond fiber source between 770-1180 nm enabled by self-phase modulation, and the wide spectral coverage is suitable for most of two-photon fluorescence microscopy applications. Based on femtosecond Yb:fiber laser, we also compared spectral broadening in different dispersion regimes using different photonic crystal fibers. We managed to maximize the self-phase modulated feature from the broadened spectra while avoiding unwanted nonlinear temporal trapping. The optimization of fiber selection and laser input conditions led to a wide tunability down to below 800nm region, which is the most commonly used two-photon excitation wavelength for many intrinsic fluorescent labels in biological tissues. We believe this fiber-based femtosecond source can be a relatively cost-effective and robust solution for most of two-photon fluorescence microscopy applications.
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