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A research team led by Takashi Saito of Ehime University Graduate School of Medicine has developed a 2-photon excitation light-sheet fluorescence microscope that (1) reduces phototoxicity, (2) expands the field of view, and (3) improves space Resolution.
When the microscope is used to observe the medaka fish, it can observe the whole embryo (expanded field of view) at the cell-level resolution (high spatial resolution) without affecting the growth of the fish (low phototoxicity). development. This result was published in the scientific journal Springer Nature.
Fluorescence microscopes are widely used in the field of life sciences to observe molecules in cells in a non-invasive manner. The light sheet fluorescence microscope can record three-dimensional images with high acquisition speed and high spatial resolution.
However, in traditional light sheet microscopes, it is difficult to limit the light damage to living tissues, and it is also difficult to simultaneously achieve a wide field of view and high (cell-level) spatial resolution.
The Ehime University research team of Takashi Saitou, Sota Takanezawa and Takeshi Imamura used the phenomenon of two-photon excitation as the key to solving this problem. The two-photon excitation microscope with infrared laser can perform gentle (low phototoxicity) imaging of living organisms.
However, since light must be focused in a narrow range to induce two-photon excitation, the excitation range (in a light sheet microscope, the field of view) is very narrow. In order to solve this problem, we have developed a simple illumination optical unit with a Bessel beam, which can extend the laser propagation range in the direction of the optical axis.
When using a 10x magnification NA0.3 objective lens, the device can stretch the beam length to 600-1000 μm while maintaining an axial resolution of 2-3 μm. Using this optical unit, we constructed a two-photon excitation light-sheet microscope, which makes it possible to image the whole body of the medaka larva with cell resolution.
Medaka is widely used as a model organism for vertebrates. Due to its small size and transparency, it is suitable for fluorescence imaging. In order to evaluate the applicability of our microscope to organisms, we conducted a phototoxicity evaluation.
Compared with the traditional Gaussian beam light sheet microscope, this shows that light damage is reduced. Therefore, it is recommended for long-term real-time imaging. Then we applied the long-term time-lapse imaging of the transgenic medaka, in which the lymphatic endothelium was labeled with green fluorescent protein, and the real-time imaging was successfully performed in three days at 5-minute intervals.
In this research, we developed a new high-performance light-sheet fluorescence microscope. Using this technology, we can observe almost all embryonic growth processes of medaka fish with high cell resolution on the entire fish body.
This technology is expected to contribute to the molecular level understanding of embryonic development, the elucidation of the pathogenesis of lifestyle-related diseases, and the further development of drug development technologies.
Takanezawa, S., etc. (2021) A wide-field light sheet microscope with two-photon Bessel beam illumination controlled by a lens axis prism. Natural communication. doi.org/10.1038/s41467-021-23249-y.
Posted in: Equipment/Technical News | Medical News
Tags: B cell, cell, embryo, embryonic development, fish, fluorescence, fluorescence microscope, fluorescent protein, imaging, life science, magnification, medicine, microscope, microscope, phototoxicity, reproduction, protein, research, T cell, genetically modified
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