Conferring optical sectioning capabilities on light field microscopy improves signal-to-noise ratio while maintaining high imaging speed.
In light field microscopy (LFM), a volumetric image can be acquired in a single snapshot, allowing high-speed imaging. The technology involves imaging the sample through an array of microlenses and computationally reconstructing the imaged volume. However, “background noise is a major limitation to further improvement of the light field performance,” says Kai Wang from the Institute of Neuroscience at the Chinese Academy of Sciences in Shanghai.
“All the current modern technologies, including confocal, two-photon, light sheet — they all use different ways to eliminate the background,” says Wang. For LFM, such background elimination has been achieved by, for example, computational approaches during image reconstruction or by light-sheet illumination. However, these techniques are limited to the imaging of neuronal activity or to samples that are amenable to illumination from the side, respectively. To image moving zebrafish or image in the scattering mouse brain, Wang and his collaborators have come up with a more generalizable strategy by marrying confocal microscopy with LFM.
Wang has been toying with ideas for reducing background signals in LFM for a while. He initially thought that combining a confocal approach with LFM might not be a good idea, and early efforts didn’t bear fruit. But he eventually came up with a strategy that worked. “You cannot use highly focused spots for excitation; you’ll need to use a very slim, long light sheet — a vertical light sheet — for excitation,” says Wang. This thin light sheet is swept across the microlenses and illuminates the sample from different angles. Fluorescence is then collected through a mask that serves as an array of apertures and that only passes light from a certain depth range. According to Wang, the key to this strategy was that “we set different apertures for different microlenses.” Since this setup limits the collection of light to a certain depth range, background noise is reduced, but so is the depth of the imaged volume. To overcome this problem, Wang combined the aperture masks with glass plates of different thicknesses, which shift the focus of the arrangement to different depths. Several of these are then mounted on a fast-rotating wheel, so that different depths can be imaged sequentially at high speed.
Wang and his collaborators used their confocal LFM to image whole-brain calcium activity in restrained zebrafish as well as in freely swimming zebrafish. They achieved single-cell resolution at an imaging speed of 6 Hz, which can be further improved by using faster and more sensitive cameras. When limiting the imaged depth range, the team could acquire data at 70 Hz, which they demonstrated by imaging circulating blood cells in the mouse brain. “We would like to combine the technology with calcium imaging to give a more complete picture of neurovascular coupling,” says Wang.
Wang plans to further develop the confocal LFM technology — for example, by optimizing the optics. “When you develop a technology, you really want to make it useful,” says Wang. It’s not just about publishing a paper. “But a major goal is trying to push the application of the technology,” says Wang.
Zhang, Z. et al. Imaging volumetric dynamics at high speed in mouse and zebrafish brain with confocal light field microscopy. Nat. Biotechnol. https://doi.org/10.1038/s41587-020-0628-7 (2020).
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Vogt, N. Volumetric imaging with confocal light field microscopy. Nat Methods 17, 956 (2020). https://doi.org/10.1038/s41592-020-00973-9