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首页> 美国卫生研究院文献>Biophysical Journal >Cooperative 4Pi Excitation and Detection Yields Sevenfold Sharper Optical Sections in Live-Cell Microscopy
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Cooperative 4Pi Excitation and Detection Yields Sevenfold Sharper Optical Sections in Live-Cell Microscopy

机译：协作4Pi激发和检测在活细胞显微镜中产生了更清晰的光学截面七倍

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摘要

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at ∼80 nm axial resolution.
机译：尽管仅在焦点处添加激发光场，或在检测器处仅添加荧光场，就足以将4Pi荧光显微镜的分辨率提高三到五倍，但通过同时利用这两种效果，可以实现其光学性能的显着改善同时。它们不仅包括光带宽的额外扩展，而且还包括新获得的空间频率到图像的放大传输。在这里，我们报告这种C型4Pi显微镜模式的实现和成像特性，该模式也是迄今为止最大光圈的远场显微镜。我们表明，结合双光子激发，所得的光学传递函数在水性样品中比共聚焦显微镜显示轴向三维分辨率提高了七倍，更重要的是，在其内部支持区域内所有频率都有明显的传递。后者也没有共焦针孔。因此，对于共聚焦和非聚焦活细胞4Pi成像，线性图像去卷积都是可能的。这种方法在最先进的扫描显微镜中实现，能够在轴向分辨率约为80 nm的情况下对固定和活细胞进行可靠的三维成像。

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