在活细胞显微镜检查中,合作式4Pi激发和检测可产生比原来清晰七倍的光学切片。
Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy.
作者信息
Gugel Hilmar, Bewersdorf Jörg, Jakobs Stefan, Engelhardt Johann, Storz Rafael, Hell Stefan W
机构信息
Leica Microsystems Heidelberg GmbH, 68165 Mannheim, Germany.
出版信息
Biophys J. 2004 Dec;87(6):4146-52. doi: 10.1529/biophysj.104.045815. Epub 2004 Sep 17.
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 approximately 80 nm axial resolution.
尽管仅在焦点处添加激发光场,或仅在探测器处添加荧光场,就足以使4Pi荧光显微镜的分辨率提高三到五倍,但同时利用这两种效应可实现其光学性能的显著改善。这些改善不仅包括光学带宽的额外扩展,还包括将新获得的空间频率放大传输到图像中。在此,我们报告这种C型4Pi显微镜模式的实现及其成像特性,它也是迄今为止孔径最大的远场显微镜。我们表明,与双光子激发相结合,所得的光学传递函数在水性样品中相对于共聚焦显微镜,轴向三维分辨率提高了七倍,更重要的是,其内部支持区域内的所有频率都有明显的传输。即使没有共聚焦针孔,后者也存在。因此,对于共聚焦和非共聚焦活细胞4Pi成像,线性图像去卷积都是可行的。在最先进的扫描显微镜中实现这一方法,能够以约80nm的轴向分辨率对固定细胞和活细胞进行稳健的三维成像。