High resolution, high speed, long working distance, large field of view confocal fluorescence microscope

Rongguang Liang,Monica K. Chawla,Chengliang Wang,Carol A. Barnes,Shaun Pacheco,Brend K. Baggett,Urs Utzinger,Minhkhoi Nguyen

doi:10.1038/s41598-017-13778-2

Rongguang Liang, Monica K. Chawla + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-13778-2

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Abstract

Confocal fluorescence microscopy is often used in brain imaging experiments, however conventional confocal microscopes are limited in their field of view, working distance, and speed for high resolution imaging. We report here the development of a novel high resolution, high speed, long working distance, and large field of view confocal fluorescence microscope (H2L2-CFM) with the capability of multi-region and multifocal imaging. To demonstrate the concept, a 0.5 numerical aperture (NA) confocal fluorescence microscope is prototyped with a 3 mm × 3 mm field of view and 12 mm working distance, an array of 9 beams is scanned over the field of view in 9 different regions to speed up the acquisition time by a factor of 9. We test this custom designed confocal fluorescence microscope for future use with brain clarification methods to image large volumes of the brain at subcellular resolution. This multi-region and multi-spot imaging method can be used in other imaging modalities, such as multiphoton microscopes, and the field of view can be extended well beyond 12 mm × 12 mm.

Highlights

Reconstructing wiring diagrams in the brain still remains a significant challenge for neuroscientists
One common method used to reconstruct wiring diagrams is to cut the whole brain into smaller sections[1,2,3]
Even though the brain clarification methods solve the problem of light penetration depth for brain imaging, microscopes are typically not optimized for whole brain imaging

Summary

System performance

One critical component to achieving whole brain activity mapping is to refine the tools available for macromolecular labelling of active cells To this end we have begun to optimize the coupling of tissue clearing methods combined with amplification of fluorescence labelling probes using hybridization chain reaction techniques. Fluorescence in situ hybridization (FISH) of the immediate early gene Arc was used to image a cleared rat brain using a conventional 2 photon microscope.

Discussion

Findings

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