We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single-and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.
Imaging across length scales and in depth has been an important pursuit of widefield optical imaging. This promises to reveal fine cellular detail within a widefield snapshot of a tissue sample. Current advances often sacrifice resolution through selective sub-sampling to provide a wide field of view in a reasonable time scale. We demonstrate a new avenue for recovering high-resolution images from sub-sampled data in light sheet microscopy using deep-learning super-resolution. We combine this with the use of a widefield Airy beam to achieve high-resolution imaging over extended fields of view and depths. We characterise our method on fluorescent beads as test targets. We then demonstrate improvements in imaging amyloid plaques in a cleared brain from a mouse model of Alzheimer’s disease, and in excised healthy and cancerous colon and breast tissues. This development can be widely applied in all forms of light sheet microscopy to provide a two-fold increase in the dynamic range of the imaged length scale. It has the potential to provide further insight into neuroscience, developmental biology, and histopathology.
We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single-and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.
We demonstrate the thermal advantages of using low repetition rate, high peak power lasers for imaging in two-photon light sheet microscopy using a Bessel light beam. We compare the use of two ultrashort pulsed lasers in such an imaging system: a high repetition rate source operating at 80 MHz and a low repetition rate source operating at 1 MHz. The low repetition rate laser requires approximately one order of magnitude lower average power than the high repetition rate source to yield the same fluorescent signal. These lasers are used to image Zebrafish larvae and record their heart rates. The data show heart rate values 30% in excess of the ground truth baseline value when imaged with the high repetition rate source due to deleterious heating, whereas the low repetition rate source yields data only a few percent above this ground truth value.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.