Confocal Light Field Microscopy: High Resolution and Signal-to-Noise Ratio Through a Confocal and 3D Tracking System

By: Katya Frazier, Nicholas Yeboah, Kayla Chien, Amadou Diallo

Modification within light field microscopy has led to the development of confocal light field microscope (LFM) designs through an addition of a confocal and 3D tracking system designed to track freely moving animals, such as zebrafish and mice. According to the study, the confocal LFM was able to pick up activity of neurons up to depths of 370 μm, and follow blood cells’ movement at 70 Hz over a volume of diameter 800 μm × thickness 150 μm and depth of up to 600 μm. The confocal LFM was adapted to capture efficiently at a higher resolution and signal-to-noise ratio than its counterpart, non-confocal light field microscope. 

From cells to atoms, the microscope has been a significant aid to the advancement of science by allowing us to explore and observe the microscopic world through lenses. Conventional technologies, such as confocal microscopy and two-photon scanning microscopy, are not fast enough for analysis of rapid volumetric dynamics when addressing vivo imaging in the brain and are limited by fluorescence saturation, while many common microscopes, including light sheet microscopes and wide-field temporal-focusing two-photon microscopes, struggle in performance when scanning deep into the mammalian brain. It is through the innovation of confocal light field microscopy that allows us to bypass these issues.

With a micro-lens array of 25 micro-lenses in a mount and varying sizes of glass plate to choose from, the confocal LFM led to a big reduction in common reconstruction artifacts and focus was not a problem. Methods, such as strategic analysis of point-spread-functions in combination with different fluorescent wavelengths of red, yellow, green, cyan, and magenta channeled through outer micro-lenses, achieved minimal aberration and obtained high lateral and axial resolution, factors that are crucial for image analysis.

Only limited by the camera’s frame rate, the high speed confocal LFM apparatuses were proven to be unrestricted by laser beam excitation and mask, prevented almost all out-of-background noise, and retained all in-focus fluorescence.

Elevated levels of imaging speed combined with epi-illumination and optical configurations lead the pathway to success in research, paving the way for rapid growth. Through confocal light field microscopy, the future of pursuing brain analysis with detailed and distinct neuronal pathways in thick tissues is possible. Large gaps in research involving live animals (e.g., zebrafish, mice) could potentially be filled with this technology, especially when it comes to behaviors, sensations, and vascular networks. 3D reconstructions are instrumental in the use of confocal light field microscopy and allow for potential groundbreaking research in areas relating to health and environment at both a global and community scale.

Citations

Zhang, Zhenkun, et al. “Imaging volumetric dynamics at high speed in mouse and zebrafish brain with confocal light field microscopy.” Nature Biotechnology, vol. 39, no. 1, 10 Aug. 2020, pp. 74–83, https://doi.org/10.1038/s41587-020-0628-7.