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首页> 外文期刊>BMC Biotechnology >Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity
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Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

机译：同时多激发多光子显微镜可提高成像灵敏度和特异性

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Background Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy ( CLSM ) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes. Results Here we describe an MPM system incorporating two, independently controlled sources of two-photon excitation whose wavelengths are adjusted to maximally excite one dye while minimally exciting the other. We report increased signal-to-noise ratios and decreased false positive emission bleed-through using this novel multiple-excitation MPM (ME-MPM) compared to conventional single-excitation MPM (SE-MPM) in a variety of multi-color imaging applications. Conclusions Similar to the tremendous gain in popularity of CLSM after the introduction of multi-color imaging, we anticipate that the ME-MPM system will further increase the popularity of MPM. In addition, ME-MPM provides an excellent tool to more rapidly design and optimize pairs of fluorescence probes for multi-color two-photon imaging, such as CFP/YFP or GFP/DsRed for CLSM .
机译：背景技术多光子显微镜（MPM）与常规的宽视野和共聚焦激光扫描显微镜（CLSM）相比，具有许多优势，可对生物样品进行成像，例如激发的3D分辨率，降低的光毒性和更深的组织成像。但是，由于常见的生物荧光团的双光子吸收（TPA）峰在很大程度上重叠，因此使MPM适应关键的多色测量提出了挑战。当前，大多数多色MPM依赖于多种染料在一个中间波长处的吸光度，这会带来诸如整个染料激发效率降低和激发效率不均等问题。结果在这里，我们描述了一个包含两个独立控制的双光子激发源的MPM系统，该光源的波长被调整为最大程度地激发一种染料，而最小程度地激发另一种染料。与传统的单激发MPM（SE-MPM）相比，在多种多色成像应用中，与传统的单激发MPM（SE-MPM）相比，我们报道使用这种新型的多激发MPM（ME-MPM）提高了信噪比并减少了假阳性发射漏气。结论与引入多色成像后CLSM的巨大普及相似，我们预计ME-MPM系统将进一步增加MPM的普及。此外，ME-MPM提供了一个出色的工具，可以更快地设计和优化用于多色双光子成像的荧光探针对，例如CLSM的CFP / YFP或GFP / DsRed。

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