The revival of light-sheet microscopy opens new possibilities for the imaging of living processes.
Optical sectioning microscopy techniques such as confocal or multiphoton microscopy have enabled three-dimensional imaging of living biological samples. But despite the many advances in this field, imaging strategies that offer high speeds, large fields of view or long-term imaging capacity are still in need to image whole cells, tissues and organisms at high resolution.
In the early 1900s, German scientists used a form of planar illumination to study colloidal solutions by scattered light imaging. Ninety years later, that work gave birth to a microscopy technique often called 'selective plane illumination microscopy' or 'fluorescence light-sheet microscopy'.
In this technique, the specimen is illuminated from the side with a thin laminar sheet of light and the emitted fluorescence is collected from above or below the sample. Confining the excitation to a very thin two-dimensional area that is progressively moved through the specimen allows for an overall reduction of phototoxicity and photobleaching, fast imaging, high-sensitivity detection and three-dimensional optical sectioning.
Light-sheet microscopy can be done in single-photon or two-photon excitation modes, and the illumination strategy can be static and spread over the entire field of view or follow a line-scanning approach.
Recent years have seen great developments in this microscopy method and increased uptake thanks to the appearance of commercial products. Its application to the imaging of developmental processes in whole living organisms amazed the scientific community several years ago, and this year its potential has been extended in several ways, including to the imaging of single living cells. Eric Betzig and colleagues used Bessel beams—very narrow non-diffracting light rays each surrounded by concentric rings of less intense light—in combination with two-photon excitation or structured illumination to image subcellular structures in living cells at high resolution (Nat. Methods 8, 417–423; 2011). And an even better resolution is possible by combining plane illumination regimes with super-resolution microscopy (Nat. Methods 8, 1047–1049; 2011).
We expect to see many more improvements in imaging speed and resolution in the coming years. There is little question that a brilliant future lies ahead for the use of sheet-based illumination to study biological processes, enabling an in-depth view of the living—no matter how big or small.
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Nature Methods (2013)