Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.
Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.
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DOI：
10.3390/s24020667
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发表时间：
2024-01-20
期刊：
影响因子：
--
通讯作者：
Verhoef AJ
中科院分区：
文献类型：
--
作者：
Fernández A;Classen A;Josyula N;Florence JT;Sokolov AV;Scully MO;Straight P;Verhoef AJ
细菌样品的固有荧光已被证明具有用于无标记细菌表征、监测细菌代谢功能以及作为跟踪相关成分通过囊泡运输的机制的潜力。多光子成像提供的激发的散射和轴向限制减少，可用于克服单光子激发（例如散射和面外光漂白）对细菌群落成像的一些限制。在这项工作中，我们使用超快 Yb 光纤激光放大器，基于蓝色内源荧光团的激发，展示了链霉菌细菌群落的体内多光子显微镜成像。其参数，如脉冲能量、持续时间、波长和重复率，可以通过同时激发不同荧光团的两光子和三光子，利用单一光源实现体内多色成像。 1040 nm 处的三光子激发可以处理具有蓝色和绿色发射光谱的荧光团（分别及其相应的紫外线和蓝色单光子激发波长），而相同波长的两光子激发则可以处理具有黄色、橙色或红色发射光谱的荧光团（及其相应的绿色、黄色和橙色单光子激发波长）。我们证明，在具有高密度荧光结构的样品中，三光子激发允许在超过 6 个有效衰减长度的深度范围内进行成像，相当于 800 微米的成像深度。
The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.
影响因子：
3.7
作者：
Willemse J;van Wezel GP
通讯作者：
van Wezel GP