The single collimating lens-based dual-beam exposure system is a holographic exposure system that is suitable for fabricating long-period gratings. The interference wavefront aberration in this system requires optimization. We analyzed the coma of a single collimating lens-based dual-beam exposure system, and the linear relationships between the coma and the positions of the pinholes and substrate are obtained. Based on these relationships, we can eliminate the coma by adjusting the pinholes or substrate along the optical axis of the collimating lens. Consequently, the defocus aberration, astigmatism, low-order spherical aberration, and coma are all simultaneously optimized by adjusting the exposure system to meet the pinhole symmetry condition and coma optimization condition. Because the optimized interference wavefront aberration includes only high-order aberrations with low peak to valley values, a high-quality exposure wavefront can be obtained. Furthermore, a 5-μm period grating exposure system is constructed with a spherical collimating lens of 0.6λ spherical aberration. Through the optimization process, a low spacing error of ∼0.04λ within an aperture of 65 × 65 mm2 is achieved (the theoretical value is 0.002λ).
Grayscale camera can only obtain gray scale image of object, while the multicolor imaging technology can obtain the color information to distinguish the sample structures which have the same shapes but in different colors. In fluorescence microscopy, the current method of multicolor imaging are flawed. Problem of these method is affecting the efficiency of fluorescence imaging, reducing the sampling rate of CCD etc. In this paper, we propose a novel multiple color fluorescence microscopy imaging method which based on the Frequency division multiplexing (FDM) technology, by modulating the excitation lights and demodulating the fluorescence signal in frequency domain. This method uses periodic functions with different frequency to modulate amplitude of each excitation lights, and then combine these beams for illumination in a fluorescence microscopy imaging system. The imaging system will detect a multicolor fluorescence image by a grayscale camera. During the data processing, the signal obtained by each pixel of the camera will be processed with discrete Fourier transform, decomposed by color in the frequency domain and then used inverse discrete Fourier transform. After using this process for signals from all of the pixels, monochrome images of each color on the image plane can be obtained and multicolor image is also acquired. Based on this method, this paper has constructed and set up a two-color fluorescence microscope system with two excitation wavelengths of 488 nm and 639 nm. By using this system to observe the linearly movement of two kinds of fluorescent microspheres, after the data processing, we obtain a two-color fluorescence dynamic video which is consistent with the original image. This experiment shows that the dynamic phenomenon of multicolor fluorescent biological samples can be generally observed by this method. Compared with the current methods, this method can obtain the image signals of each color at the same time, and the color video’s frame rate is consistent with the frame rate of the camera. The optical system is simpler and does not need extra color separation element. In addition, this method has a good filtering effect on the ambient light or other light signals which are not affected by the modulation process.
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