ABERRATIONS IN CONFOCAL FLUORESCENCE MICROSCOPY INDUCED BY MISMATCHES IN REFRACTIVE-INDEX
ABERRATIONS IN CONFOCAL FLUORESCENCE MICROSCOPY INDUCED BY MISMATCHES IN REFRACTIVE-INDEX
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DOI：
10.1111/j.1365-2818.1993.tb03315.x
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发表时间：
1993-03-01
影响因子：
2
通讯作者：
STELZER, EHK
中科院分区：
文献类型：
--
作者：
HELL, S;REINER, G;STELZER, EHK
从理论上研究了在生物样品观察中折射率失配对共焦荧光显微镜图像采集过程的影响。该分析考虑了光的矢量特性，适用于高数值孔径。对实际情况下的分辨率下降、光强下降和焦移等问题给出了定量预测。当用1.3的数值孔径（油浸）和514 nm的激发波长观察时，焦点的中心在水性介质中每10 μ m的轴向位移移位1.7 μ m，从而产生在轴向方向上按1.2的因子缩放的图像。此外，可以预期，对于水性介质内20 μ m深的荧光平面，峰强度比10 μ m深的平面小40%。此外，轴向分辨率降低了1.4倍。对不同折射率的测试样品进行了实验验证。
The effect of refractive-index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1.3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1.7 mum per 10 mum of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1.2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 mum deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 mum deep. In addition, the axial resolution is decreased by a factor of 1.4. The theory was experimentally verified for test samples with different refractive indices.