Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes.
Addressing the autofluorescence issue in deep tissue imaging by two-photon microscopy: the significance of far-red emitting dyes.
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DOI：
10.1039/c7sc03362a
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发表时间：
2017-11-01
期刊：
影响因子：
8.4
通讯作者：
Ahn KH
中科院分区：
文献类型：
--
作者：
Jun YW;Kim HR;Reo YJ;Dai M;Ahn KH
我们已经开发出一种新的双光子吸收染料，它是远红色的，水溶性的，在细胞和组织内都非常明亮。在组织成像中，来自黄色波长区域的显著自身荧光可以通过深红色发射染料来解决。组织的荧光成像对于研究细胞水平以外的生物事件是必不可少的。基于荧光染料非线性光吸收的双光子显微镜是一种可行的高分辨率组织成像工具。深层组织成像的一个关键限制是来自内在生物分子的自身荧光。在这里，我们报告了一个系统的研究，揭示了相对的自体荧光干涉，这是依赖于组织的类型和激发和发射波长在双光子成像。在小鼠的脑、肾、肝、肺和脾组织中，肾脏组织表现出明显的自身荧光，其次是肝脏和其他组织。值得注意的是，无论组织类型如何，不仅在绿色发射通道中观察到明显的自身荧光，而且在黄色发射通道中也观察到明显的自身荧光，其中常见的双光子吸收染料也会发射，而在红色通道中观察到最小的自身荧光。激发波长对自身荧光有轻微的影响。为了减少自身荧光，我们开发了一类新的双光子吸收染料，这种染料是远红色的，水溶性的，在细胞和组织内都很亮。对成像深度的比较评估，这取决于在蓝绿、黄和远红区域发射的三种选定的染料，显示了远红发射染料对深部组织成像的重要性。
We have 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. The significant autofluorescence from yellow wavelength region in tissue imaging can be addressed by deep-red emitting dyes. 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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DOI：
10.1073/pnas.95.26.15741
发表时间：
1998-12-22
影响因子：
11.1
作者：
Kleinfeld, D;Mitra, PP;Denk, W
通讯作者：
Denk, W
影响因子：
3.5
作者：
Durr, Nicholas J.;Weisspfennig, Christian T.;Ben-Yakar, Adela
通讯作者：
Ben-Yakar, Adela
影响因子：
3.5
作者：
Kobat, Demirhan;Horton, Nicholas G.;Xu, Chris
通讯作者：
Xu, Chris
影响因子：
3.8
作者：
Kobat, Demirhan;Durst, Michael E.;Xu, Chris
通讯作者：
Xu, Chris
影响因子：
15
作者：
Kim, Dokyoung;Moon, Hyunsoo;Ahn, Kyo Han
通讯作者：
Ahn, Kyo Han