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Proceedings Paper

Modulation of the pupil function of microscope objective lens for multifocal multi-photon microscopy using a spatial light modulator
Author(s): Naoya Matsumoto; Shigetoshi Okazaki; Hisayoshi Takamoto; Takashi Inoue; Susumu Terakawa
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Paper Abstract

We propose a method for high precision modulation of the pupil function of a microscope objective lens to improve the performance of multifocal multi-photon microscopy (MMM). To modulate the pupil function, we adopt a spatial light modulator (SLM) and place it at the conjugate position of the objective lens. The SLM can generate an arbitrary number of spots to excite the multiple fluorescence spots (MFS) at the desired positions and intensities by applying an appropriate computer-generated hologram (CGH). This flexibility allows us to control the MFS according to the photobleaching level of a fluorescent protein and phototoxicity of a specimen. However, when a large number of excitation spots are generated, the intensity distribution of the MFS is significantly different from the one originally designed due to misalignment of the optical setup and characteristics of the SLM. As a result, the image of a specimen obtained using laser scanning for the MFS has block noise segments because the SLM could not generate a uniform MFS. To improve the intensity distribution of the MFS, we adaptively redesigned the CGH based on the observed MFS. We experimentally demonstrate an improvement in the uniformity of a 10 × 10 MFS grid using a dye solution. The simplicity of the proposed method will allow it to be applied for calibration of MMM before observing living tissue. After the MMM calibration, we performed laser scanning with two-photon excitation to observe a real specimen without detecting block noise segments.

Paper Details

Date Published: 28 February 2014
PDF: 8 pages
Proc. SPIE 8948, Multiphoton Microscopy in the Biomedical Sciences XIV, 89481Q (28 February 2014); doi: 10.1117/12.2038929
Show Author Affiliations
Naoya Matsumoto, Hamamatsu Photonics K.K. (Japan)
Shigetoshi Okazaki, Hamamatsu Univ. School of Medicine (Japan)
Hisayoshi Takamoto, Hamamatsu Photonics K.K. (Japan)
Takashi Inoue, Hamamatsu Photonics K.K. (Japan)
Susumu Terakawa, Hamamatsu Univ. School of Medicine (Japan)


Published in SPIE Proceedings Vol. 8948:
Multiphoton Microscopy in the Biomedical Sciences XIV
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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