Advancing Differential Confocal Microscopy with ITORDM: Overcoming Axial Measurement Limitations

Significance 

The field of microscopy has seen significant advancements with confocal microscopic (CM) imaging techniques becoming a vital tool in various applications such as in aerospace, biomedical sciences, and semiconductor technology. CM techniques are recognized for their high contrast, exceptional resolution, and the ability to provide detailed three-dimensional (3D) images. However, traditional CM techniques are constrained by the spatial conjugation of their double pinholes, necessitating point-by-point or layer-by-layer scanning for 2D and 3D imaging, respectively. The accuracy and efficiency of axial measurements are limited by the scanning step and peak extraction algorithms.

To enhance the efficiency and accuracy of axial measurements, researchers have proposed various differential confocal axial measurement methods. One such method, image-squared differential confocal microscopic measurement, which effectively doubled the sensitivity compared to traditional CM methods. Further enhancements and optimizations were introduced. However, these methods faced limitations in terms of axial measurement range and potential misjudgments of axial positions, especially for free-form complex shaped surfaces. Moreover, these methods assumed that the measured differential signal always fell within the effective range of the pre-calibrated differential axial response curve (DARC) between the differential intensity signal and axial position. This assumption is often invalid for free-form complex-shaped surfaces, and in nano-microscopic measurements, it is challenging to know the surface height information of the sample in advance. This leads to a pressing need for a method that determines the validity of measurement for each point within the field of view and addresses the challenge of differential confocal axial effective measurement over the range.

In response to these challenges, a new study published in the Applied Optics led by Dr. Tao Yuan, Dr. Dingrong Yi, Dr. Yiqing Ye, Dr. Dongliang Wu, Dr. Wei Jiang, Dr. Ting Liu from the Huaqiao University introduced a novel approach called the Information Theory-based Differential Confocal Over-Range Determination Method (IT-ORDM). This new method aims to determine the effective measurement range of differential confocal axial measurement through an information theory-based analysis of the axial light intensity response curves. The IT-ORDM addressed the limitations of existing methods and significantly improved the determination of effective measurement areas for differential confocal measurement. Moreover, the new method leverages the out-of-focus characteristics of the differential confocal axial light intensity response curve, coupled with the confocal axial light intensity response curve. It begins by locating the boundary position of the axial effective measurement range using DARC. Subsequently, it determines the effective intensity measurement ranges for the pre-focus and post-focus Axial Response Curves (ARCs). Finally, it employs an intersection operation to extract the effective measurement area of differential confocal measurement. The results of the experimental verification demonstrate that the IT-ORDM successfully resolves the problem of determining the effective area of differential confocal axial measurement, paving the way for pixel segmentation and 3D restoration within the effective measurement range. The IT-ORDM first identified the boundary positions of the axial effective measurement range using the DARC. It then determined the effective intensity measurement ranges of both pre-focus and post-focus ARCs. By intersecting the effective measurement ranges of pre-focus and post-focus, the IT-ORDM accurately extracts the effective measurement area for differential confocal measurements.

The authors presented a series of experimental results to validate the efficacy of IT-ORDM. Using a multi-stage sample with gauge blocks, the study demonstrates how the method determines the grayscale values corresponding to the effective measurement interval, subsequently identifying the effective measurement area and performing 3D measurements. They demonstrated the effectiveness of the IT-ORDM in determining the valid measurement area for differential confocal axial measurement and achieving accurate 3D restoration within this range. The proposed method has significant potential in improving the reliability and accuracy of 3D imaging using differential confocal microscopy. In conclusion, IT-ORDM represents a significant advancement in the field of differential confocal microscopy. It addresses the limitations of existing methods by introducing a novel approach to determine the axial over-range of measurement. By leveraging information theory and the out-of-focus characteristics of the differential confocal axial light intensity response curve, IT-ORDM offers a robust solution to a long-standing problem. According to the authors, the IT-ORDM method provides a means to accurately assess the effectiveness of measurement for each point within the field of view, thereby ensuring the authenticity of data in the measurement process. Moreover, the method effectively overcomes the challenges posed by free-form complex-shaped surfaces and the lack of prior knowledge about sample height information in nano-microscopic measurements. The new approach by Huaqiao University  scientists opens the door to more intelligent and accurate differential confocal microscopy techniques, ultimately enhancing the capabilities of this crucial imaging technology.

Reference

Yuan T, Yi D, Ye Y, Wu D, Jiang W, Liu T. Differential confocal over-range determination method based on an information theory. Appl Opt. 2023;62(8):2073-2077. doi: 10.1364/AO.484018.

Go to Appl Opt.

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