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.
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. [APBN]
Source: Takanezawa et al. (2021). Wide field light-sheet microscopy with lensaxicon controlled two-photon Bessel beam illumination. Nature communications, 12(1), 1-15.