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Addressing the autofluorescence issue in deep tissue imaging by two-photon
microscopy: the significance of far-red emitting dyes
#MMPMID29568432
Jun YW
; Kim HR
; Reo YJ
; Dai M
; Ahn KH
Chem Sci
2017[Nov]; 8
(11
): 7696-7704
PMID29568432
show ga
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.
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