Researchers from Ehime University Graduate School of Medicine have developed a two-photon excitation light-sheet fluorescence microscope, offering an extended field of vision, with superb spatial resolution, and reduced phototoxicity for live imaging of biological samples.
A ubiquitous laboratory staple for most biologists, fluorescence microscopes allow researchers to take a closer look at cell samples in a non-invasive manner by generating images using fluorescence. This versatile tool is similar to conventional light microscopes, but with additional features like being able to visually enhance three-dimensional features at small scales.
One particular type of fluorescence microscope is the light-sheet fluorescence microscope which enables the recording of three-dimensional snapshots with high acquisition speed and remarkable spatial resolution. However, its technical prowess does not come without a price. In comparison to its predecessors – the conventional light-sheet microscopes – its fluorescence poses significant photodamage to living tissues, thus complicating the means to achieve a wide field of vision and high cell-level resolution. Fortunately, researchers have finally found the key to enjoy the best of all three worlds - a wide field of vision, excellent resolution, and low toxicity.
A group of researchers led by Takashi Saito of the Ehime University Graduate School of Medicine have recently created a two-photon excitation light-sheet fluorescence microscope, which boasts reduced phototoxicity, expanded field of vision and increased spatial resolution. By manipulating the two-photon excitation phenomena, the team was able to create a microscope with gentle lasers that pose minimal phototoxic effects on living samples.
However, with bold innovations come great challenges. In exchange for reducing photodamage with two-photon excitation, the team was limited to focusing their light on an incredibly narrow excitation range. To overcome this hurdle, the researchers came up with a novel solution of developing an illumination optics unit with a Bessel beam. In doing so, they managed to lengthen the laser propagation range in the direction of the optic axis. This unit effectively extended the beam length to 600-1000 μm while sustaining a 2-3 μm axial resolution when tested under 10x magnification. With this optical unit, they were able to successfully craft their two-photon excitation light-sheet microscopy.
Following its development, the microscope was evaluated on its performance and applicability. The team used the microscope to inspect a medaka fish embryo, which is a model organism for vertebrates. Not only was it possible to view the whole body of the embryo at a cellular level resolution, but the image was also made perceptible without compromising the growth of the fish, validating its low phototoxicity effects.
When the results were compared with a conventional Gaussian beam light-sheet microscope, the image obtained from the two-photon microscope demonstrated significant reductions in photodamage, hence suggesting its potential use for long-term live imaging. The team then attempted a prolonged observation of the transgenic fish through time-lapse imaging every five minutes over three days and succeeded in live imaging the green fluorescent-stained lymphatic endothelium of transgenic medaka.
This high-performance light-sheet fluorescence microscope is foreseen to open doors to discoveries and inventions needed to help us unravel the mysteries that remain unexplored in science, from achieving a better understanding of the molecular mechanisms taking place during embryonic development and disease pathogenesis to advancing drug development.
Source: Takanezawa et al. (2021). Wide field light-sheet microscopy with lensaxicon controlled two-photon Bessel beam illumination. Nature communications, 12(1), 1-15.