Issue 11, 2017

Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes

Abstract

The fluorescence imaging of tissue is essential for studying biological events beyond the cellular level. Two-photon microscopy based on the nonlinear light absorption of fluorescent dyes is a viable tool for the high resolution imaging of tissue. A key limitation for deep tissue imaging is the autofluorescence from intrinsic biomolecules. Here, we report a systematic study that discloses relative autofluorescence interference, which is dependent on the type of tissue and the excitation and emission wavelengths in two-photon imaging. Among the brain, kidney, liver, lung, and spleen mouse tissues examined, the kidney tissue exhibited prominent autofluorescence followed by the liver and others. Notably, regardless of the tissue type, prominent autofluorescence is observed not only from the green emission channel but also from the yellow emission channel where common two-photon absorbing dyes also emit, whereas there is minimal autofluorescence from the red channel. The autofluorescence is slightly influenced by the excitation wavelength. Toward minimal autofluorescence, we developed a new class of two-photon absorbing dyes that are far-red emitting, water-soluble, and very bright inside cells as well as in tissue. A comparative assessment of the imaging depth, which is dependent on the three selected dyes that emit in the blue-green, yellow, and far-red regions, shows the importance of far-red emitting dyes for deep tissue imaging.

Graphical abstract: Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes

Supplementary files

Article information

Article type
Edge Article
Submitted
02 Aug 2017
Accepted
15 Sep 2017
First published
18 Sep 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 7696-7704

Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes

Y. W. Jun, H. R. Kim, Y. J. Reo, M. Dai and K. H. Ahn, Chem. Sci., 2017, 8, 7696 DOI: 10.1039/C7SC03362A

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