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.

中文翻译：

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共聚焦激光扫描显微镜的衰减校正及其在色谱中的应用

共聚焦激光扫描显微镜的适用性受到限制，例如受到激发和荧光发射光束的衰减。作为对获得的显微图像进行进一步处理和分析的先决条件，提出了一种校正这种衰减的新方法。校正基于光束建模和修正比尔-朗伯定律的微分形式。事实证明，强度衰减可以建模为显微图像与激发半光束和发射光束的强度的双重卷积。在对荧光辐射强度和检测信号进行弱假设的情况下，推导出衰减校正和衰减模拟的公式。该方法可追溯到 Roerdink 发表的方法，该方法针对更真实的光束建模进行了修改，避免了所谓的弱衰减扩展，并考虑了整个激发光束光锥的荧光激发。该方法的适用性在合成实例以及色谱珠的显微图像中得到了证明。结果表明，即使显微图像受到强衰减的影响，新方法也可以成功应用于重建样本中真实的荧光团分布。