Light-sheet fluorescence microscopy allows researchers to create 3D images of tissues and live animal embryos using fluorescent tags. As reported in the journal Optics Letters, three-photon absorption can be used with light-sheet fluorescence microscopy to image deeper into tissues. As a demonstration, they used the combined technique to produce clear images throughout a ball of cultured cells, known as a spheroid, about 450 microns in diameter.
This demonstration provided better imaging at depth, helping scientists gain better data about biological processes. Team leader, Kishan Dholakia of University of St. Andrews in the U.K. said “This approach could be especially useful for neuroscience and developmental biology studies and might find application in imaging multiple samples in an automated way for drug discovery.”
To further improve the depth imaging and field of view, the researchers experimented with changing the light intensity profile of the laser to a Bessel beam, which has a central bright core surrounded by concentric rings, rather than the traditional solid Gaussian laser beam like that of a laser pointer.
“Bessel beams can be used in two-photon light-sheet mode but may yield potential artifacts due to their concentric rings,” said co-author Federico Gasparoli. “For the first time, we show numerically and experimentally that these problems are suppressed in three-photon light-sheet fluorescence microscopy and that the beam goes even deeper, making this approach very attractive.”