Design, assembly, and metrology of an oil-immersion microscope objective with long working distance

Wei-Jei Peng; Wen-Lung Lin; Hui-Jean Kuo; Cheng-Fang Ho; Wei-Yao Hsu

doi:10.1117/12.2246254

31 October 2016 Design, assembly, and metrology of an oil-immersion microscope objective with long working distance

Wei-Jei Peng, Wen-Lung Lin, Hui-Jean Kuo, Cheng-Fang Ho, Wei-Yao Hsu

Proceedings Volume 10021, Optical Design and Testing VII; 100211N (2016) https://doi.org/10.1117/12.2246254
Event: SPIE/COS Photonics Asia, 2016, Beijing, China

Abstract

The design, tolerance sensitivity reduction, assembly, and optical bench test for an oil-immersion microscope objective with long working distance employed in a lattice light-sheet microscope is presented in this paper. In this application, the orthogonal excitation and detection objectives are dipped in an oil medium. The excitation objective focuses the incident laser beam to generate fluorescence on specimen for collecting by detection objective. The excitation objective is custom-designed to meet the requirement specification such as oil-immersion, the long working distance, and numerical aperture (NA) of 0.5, etc. To produce an acceptable point spread function (PSF) for effective excitation, the performance of the objective needs to be close to diffraction limit. Because the tolerance of the modulation transfer function (MTF) is more and more sensitive at higher spatial frequency, it is extremely critical to keep the performance after manufacture. Consequently, an insensitive optical design is very important for relaxing tolerance. We compare the design with and without tolerance sensitivity reduction, and the as-built MTF shows the result. Furthermore, the method for sensitivity reduction is presented. The opto-mechanical design and assembly method are also discussed. Eventually, the objective with five spherical lenses was fabricated. In optical bench test, the depth of the oil is sensitive to MTF, and it leads to the complicated adjustment. For solving this issue, we made an index-matching lens to replace oil for measurement easily. Finally, the measured MTF of the excitation objective can accomplish the requirement specification and successfully employed in a lattice light-sheet microscope.

Citation Download Citation

Wei-Jei Peng, Wen-Lung Lin, Hui-Jean Kuo, Cheng-Fang Ho, and Wei-Yao Hsu "Design, assembly, and metrology of an oil-immersion microscope objective with long working distance", Proc. SPIE 10021, Optical Design and Testing VII, 100211N (31 October 2016); https://doi.org/10.1117/12.2246254

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KEYWORDS

Modulation transfer functions

Objectives

Tolerancing

Optical design

Spatial frequencies

Microscopes

Point spread functions

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