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J Biophotonics. 2019 Jan;12(1):e201800136. doi: 10.1002/jbio.201800136. Epub 2018 Aug 30.

Saturated two-photon excitation fluorescence microscopy for the visualization of cerebral neural networks at millimeters deep depth.

Author information

1
Department of Electrical Engineering and Graduate Institute of Photonics and Optoelectronics, National Taiwan University, Taipei, Taiwan.
2
Department of Life Sciences, National Taiwan University, Taipei, Taiwan.
3
Institute of Physics and Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan.
4
Molecular Imaging Center and Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan.

Abstract

Optical imaging is a key modality for observing biological specimen with higher spatial resolution. However, scattering and absorption of light in tissues are inherent barriers in maximizing imaging depth in biological tissues. To achieve this goal, use of light at near-infrared spectrum can improve the present situation. Here, the capability of saturated two-photon saturated excitation (TP-SAX) fluorescence microscopy to image at depths of >2.0 mm, with submicron resolution in transparent mouse brain imaging, is demonstrated. At such depths with scattering-enlarged point spread function (PSF), we find that TP-SAX is capable to provide spatial resolution improvement compared to its corresponding TPFM, which is on the other hand already providing a much improved resolution compared with single-photon confocal fluorescence microscopy. With the capability to further improve spatial resolution at such deep depth with scattering-enlarged PSF, TP-SAX can be used for exquisite visualization of delicate cerebral neural structure in the scattering regime with a submicron spatial resolution inside intact mouse brain.

KEYWORDS:

brain imaging; nonlinear microscopy; saturated fluorescence excitation microscopy; two-photon fluorescence microscopy

PMID:
30112801
DOI:
10.1002/jbio.201800136

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