Department of Mathematics and Natural Sciences, University of Applied Sciences, Darmstadt, Germany.
Leica Microsystems CMS GmbH, Mannheim, Germany.
J Microsc. 2020 May;278(2):76-88. doi: 10.1111/jmi.12888. Epub 2020 Mar 22.
The applicability of confocal laser scanning microscopy is limited, e.g. by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on beam modelling and on a differential form of the modified Beer-Lambert law. It turns out that the intensity decay can be modelled as a double convolution of the microscopic image with the intensities of the excitation semibeam and the emission beam. Under weak assumptions made for the intensities of the fluorescent radiation and the detected signal, formulas for the attenuation correction and the attenuation simulation are derived. The method traces back to that one published by Roerdink which is modified concerning a more realistic beam modelling, avoiding the so-called weak attenuation expansion and considering fluorescence excitation throughout the light cone of the excitation beam. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation. LAY DESCRIPTION: The applicability of confocal laser scanning microscopy is limited by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on modeling the excitation as well as the emission beam and on a modified Beer-Lambert law for beam attenuation. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.
共聚焦激光扫描显微镜的适用性受到限制，例如激发光和荧光发射光束的衰减。作为进一步处理和分析所获得的微观图像的前提，提出了一种新的方法来校正这种衰减。该校正方法基于对激发和发射光束的建模以及对改进后的 Beer-Lambert 定律的微分形式。结果表明，强度衰减可以用微观图像与激发半光束和发射光束的强度的双重卷积来建模。在对荧光辐射和检测信号的强度做出弱假设的前提下，导出了衰减校正和衰减模拟的公式。该方法追溯到 Roerdink 发表的方法，该方法在更现实的光束建模方面进行了修改，避免了所谓的弱衰减展开，并考虑了整个激发光束光锥内的荧光激发。该方法的适用性通过合成示例以及色谱珠的微观图像得到了证明。结果表明，即使微观图像受到强烈衰减的影响，该新方法也可以成功地应用于重建标本中的真实荧光团分布。
