Super-multiplex vibrational imaging.
Super-multiplex vibrational imaging.
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DOI：
10.1038/nature22051
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发表时间：
2017-04-27
期刊：
影响因子：
64.8
通讯作者：
Min W
中科院分区：
文献类型：
--
作者：
Wei L;Chen Z;Shi L;Long R;Anzalone AV;Zhang L;Hu F;Yuste R;Cornish VW;Min W
直接可视化细胞内大量不同分子种类的能力对于理解复杂系统和过程越来越重要。尽管现有的方法已成功地用于探索神经系统中的结构-功能关系，原位RNA谱，揭示肿瘤微环境异质性或研究动态大分子组装，但在生物条件下以高选择性和灵敏度成像许多物种仍然具有挑战性。例如，荧光显微镜面临着一个“色障”，这是由于荧光光谱的固有宽（~1500 cm−1）和无特征的性质，将可分辨的颜色限制在2到5种（或7-9种，如果使用复杂的仪器和分析）。自发拉曼显微镜探测具有窄得多的共振（峰宽~10 cm−1）的振动跃迁，因此不会遇到这个问题，但其微弱的信号使许多要求苛刻的生物成像应用成为不可能。虽然表面增强拉曼散射提供了显着的灵敏度和多样性，它不能很容易地用于定量成像活细胞内的特定分子目标。在这里，我们表明，在电子预共振条件下进行受激拉曼散射（epr-SRS），使成像具有精致的振动选择性和灵敏度（低至250纳米，1毫秒）在活细胞。我们还创建了一个调色板的三键共轭近红外染料，每个显示一个单一的epr-SRS峰在细胞沉默的光谱窗口，并与可用的荧光探针给出24个可分辨的颜色与进一步扩展的潜力。对神经元共培养物和脑组织的原理验证实验揭示了在生理和病理条件下DNA和蛋白质代谢的细胞类型依赖性异质性，强调了这种超级多重光学成像方法用于解开复杂生物系统中错综复杂的相互作用的潜力。
The ability to directly visualize a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ, reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm−1) and featureless nature of fluorescence spectra that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm−1) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible. And while surface-enhanced Raman scattering offers remarkable sensitivity and multiplicity, it cannot be readily used to quantitatively image specific molecular targets inside live cells. Here we show that carrying out stimulated Raman scattering under electronic pre-resonance conditions (epr-SRS) enables imaging with exquisite vibrational selectivity and sensitivity (down to 250 nM with 1-ms) in living cells. We also create a palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems.
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DOI：
10.1126/science.1168683
发表时间：
2009-05-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Shu X;Royant A;Lin MZ;Aguilera TA;Lev-Ram V;Steinbach PA;Tsien RY
通讯作者：
Tsien RY
影响因子：
7.2
作者：
Chen, Bryan;Retzlaff, Marco;Frydman, Judith
通讯作者：
Frydman, Judith
影响因子：
13.6
作者：
Liao CS;Wang P;Wang P;Li J;Lee HJ;Eakins G;Cheng JX
通讯作者：
Cheng JX
影响因子：
48
作者：
Giesen, Charlotte;Wang, Hao A. O.;Bodenmiller, Bernd
通讯作者：
Bodenmiller, Bernd
影响因子：
82.9
作者：
Kaushik, Susmita;Cuervo, Ana Maria
通讯作者：
Cuervo, Ana Maria