【JACS】基于生物正交化学的单染料、免转染荧光寿命显微成像多重化技术（实现活细胞三结构单通道清晰区分）
【JACS】基于生物正交化学的单染料、免转染荧光寿命显微成像多重化技术（实现活细胞三结构单通道清晰区分）
文章标题：Single-Dye, Transfection-Free FLIM Multiplexing via Bioorthogonal Chemistry 文章作者：Neville Dadina # ; Justin H. Kwon # ; Lauren Lesiak ; Shuai Zheng ; Tvisha Singh ; Madeline Zoltek ; Daniel Brauer ; Alanna Schepartz * 文章链接：https://doi.org/10.1021/jacs.5c22980
研究动因：传统多靶点成像的技术瓶颈与免转染荧光寿命多重化的提出
多靶点荧光成像在活细胞生物学研究中至关重要，目前主流策略依赖于荧光蛋白或自标记蛋白标签（如HaloTag、SNAP-tag）结合多色荧光团。然而，可见光谱的重叠严重限制了同时成像的靶标数量，且依赖基因转染与工程改造的方法难以应用于原代细胞、患者来源类器官和完整组织，长时程成像也易受光毒性干扰。荧光寿命显微成像（FLIM）能够打破光谱重叠限制，在单一光谱通道内实现多重化，但以往策略大多需合成多种复杂染料或仍依赖基因工程标签。为此，本文旨在解决无需基因改造前提下的单光谱窗口多靶点成像难题，开发基于小分子生物正交反应的单染料荧光寿命多重化策略（FLIMINGO），为原代体系和活细胞的多靶标精准探测提供高效、低毒的全新工具。
Fig.1 Fluorescence turn-on correlates with fluorescence lifetime. (a) Fluorescence turn-on correlates with fluorescence lifetime in vitro. HD555 and the products of its reaction with BCN, TCO (equatorial), and SpH-containing partners along with (b) their average amplitude-weighted lifetimes determined in vitro. Adjusted P values for HD555-BCN vs HD555-TCO (****P < 0.0001), HD555-BCN vs HD555-SpH (**P = 0.0024), and HD555-TCO vs HD555-SpH (**P = 0.0012) calculated using Dunnett’s T3 multiple comparisons test. Statistical significance was determined via Welch’s and Brown-Forsythe ANOVA with post hoc Dunnett’s T3 test accounting for heteroscedasticity. (c–h) Fluorescence turn-on correlates with fluorescence lifetime in cellulo. (c) HeLa cells expressing Sec61β-HaloTag were treated with the HTL-TCO or HTL-BCN adapter shown followed by HD555 and imaged by detecting (d) fluorescence intensity (Intensity), average photon arrival time (Fast FLIM, color-coded) or using phasor analysis (Phasor). (e) Average intensity-weighted lifetimes of cells expressing the indicated organelle marker and treated with the indicated HTL-BCN or HTL-TCO adapter followed by HD555. ****P < 0.0001 for all pairwise comparisons between BCN and TCO with Sec61β-HT, TOMM20-HT, ST-Sec61β, or COX8A-ST: Sec61β-HT (Δ = 0.240 ns), TOMM20-HT (Δ = 0.226 ns), ST-Sec61β (Δ = 0.433 ns), and COX8A-ST (Δ = 0.408 ns). Adjusted P values calculated using Dunnett’s T3 multiple comparisons test. Statistical significance was determined via Welch’s and Brown-Forsythe ANOVA with post hoc Dunnett’s T3 test accounting for heteroscedasticity. (f) Individual and overlaid phasor plots of FLIM data. (g) Two-species FLIM imaging of HeLa cells expressing the indicated organelle markers (TOMM20-HaloTag and Sec61β-SNAPTag) and treated with HTL-BCN and BG-TCO or (h) HTL-TCO and BG-BCN then HD555. Scale bars, 5 μm.
研究实施方案与关键实验结果：反应调控寿命机制及活细胞多重成像验证
该研究基于荧光增强倍数、荧光量子产率与荧光寿命之间的第一性原理相关性，提出了利用单一四嗪猝灭荧光染料（HD555）与不同生物正交反应物原位发生反电子需求狄尔斯-阿尔德（IEDDA）反应的调控框架。本文首次提出的核心创新机制在于：同一四嗪染料母体在与不同亲双烯体反应时，由于反应产物结构与能量转移路径的差异，会产生具有相同发射波长但荧光寿命显著不同的产物；结合具有细胞器靶向能力的小分子探针，即可在同一光谱通道内利用相量分析（Phasor Analysis）解析不同结构。该方案改进了传统的IEDDA四嗪连接反应与相量分析技术，构建了“小分子靶向递送 原位生物正交反应 寿命区分与相量解析”的闭环技术路线。
实验首先在体外和基因标记模型（HeLa细胞表达内质网和线粒体标记物）中验证了寿命调控的可行性：体外反应中，HD555与双环壬炔（BCN）、反式环辛烯（TCO）及螺己烯反应产物呈现出极高的寿命区分度（P < 0.0001）。在免转染的活细胞实验中，研究人员合成了分别靶向线粒体内膜（HAO）、细胞核（Hoechst）和微管网络（多西他赛）的BCN与TCO偶联探针。结果表明，HAO-BCN与HAO-TCO处理细胞后的平均强度加权荧光寿命分别为 2.819 ± 0.086 纳秒 和 2.226 ± 0.078 纳秒，二者寿命差达 0.593 纳秒（P < 0.0001），相比基因标签模型（内质网寿命差 0.240 纳秒、线粒体寿命差 0.226 纳秒）展现出更强的微环境敏感性与区分度；多西他赛衍生物的寿命差更达到 0.8504 纳秒。
最终，在活体HeLa细胞中，研究人员将靶向细胞核的Hoechst-BCN、线粒体的HAO-BCN以及微管的Docetaxel-TCO同时递送至细胞内，随后加入单一染料HD555进行原位标记。利用快速FLIM采集光子到达时间并进行相量图分离，在完全不进行基因转染且仅使用单一荧光通道的条件下，成功独立解析并重构出细胞核、线粒体和微管 3种亚细胞结构 的清晰多重成像图像。
Fig.2 Single-species FLIM using multiple organelle- or structure-selective small molecules and one fluorophore. (a) TCO and BCN derivatives of HAO, Hoechst, and Docetaxel, which localize to the inner mitochondrial membrane (IMM), nucleus, or microtubule network, respectively. (b) HeLa cells were treated with HAO-BCN or HAO-TCO and (c) imaged by detecting fluorescence intensity (Intensity), average photon arrival time (Fast FLIM), or phasor analysis (Phasor). (d) Plot of average intensity-weighted fluorescence lifetimes associated with cells treated with the indicated small molecules. ****P < 0.0001 for all pairwise comparisons between BCN and TCO small molecule derivatives: HAO (Δ = 0.5930 ns), Hoechst (Δ = 0.4853 ns), and Docetaxel (Δ = 0.8504 ns). Adjusted P values calculated using Dunnett’s T3 multiple comparisons test. Statistical significance was determined via Welch’s and Brown-Forsythe ANOVA with post hoc Dunnett’s T3 test accounting for heteroscedasticity. (e) Composite phasor plot of FLIM data acquired from cells treated with HAO-BCN and HAO-TCO. (f, g) HeLa cells were treated with (f) Hoechst-BCN or Hoechst-TCO or (g) Docetaxel-BCN or Docetaxel-TCO and imaged by detecting fluorescence intensity (intensity), average photon arrival time (Fast FLIM), or phasor analysis (Phasor). Scale bars, 5 μm.
Fig.3 Two- and three-species FLIM multiplexing in a live cell using a single dye and only cell-permeant localization probes. HeLa cells were incubated with (a) Hoechst-BCN and HAO-TCO; (b) HAO-BCN and Hoechst-TCO; and (c) Hoechst-BCN and Docetaxel-TCO followed by HD555. Cells were imaged by detecting total fluorescence intensity (Intensity) or average photon arrival time (Fast FLIM, color-coded) or using phasor analysis to generate the single-species and dual-species images shown. (d) HeLa cells were incubated with Hoechst-BCN, HAO-BCN, and Docetaxel-TCO. Scale bars, 5 μm.
核心创新与性能提升：单荧光分子破局多靶点活细胞成像
本文的核心突破在于确立了利用单一荧光染料与单一生物正交反应体系实现免转染活细胞多靶点成像（FLIMINGO） 的新范式，直接攻克了传统多重成像中光谱拥挤、合成复杂及高度依赖基因工程转染的核心痛点。实验证明，该方法利用反应物依赖性及亚细胞微环境对寿命的调制作用，实现了高达 0.85 纳秒 的寿命差异分离，在单一光谱窗口内达成了3个独立细胞结构的高对比度无干扰成像，并验证了TAMRA-四嗪等常规染料的通用性。作者同时指出，部分超快亲双烯体（如螺己烯基团）在细胞内存在稳定性限制，未来进一步开发高稳定性反应底物与拓展近红外波段荧光染料将是该技术深化应用的重要方向。
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