Maruyama Atsushi, Oshima Yusuke, Kajiura-Kobayashi Hiroko, Nonaka Shigenori, Imamura Takeshi, Naruse Kiyoshi
Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki, Aichi, 444-8585, Japan ; These authors contributed equally to this work.
Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Division of Bio-imaging, Proteo-Science Center, Ehime University, Shitukawa Toon city, Ehime, 791-0295, Japan ; Translational Research Center, Ehime University Hospital, Shitukawa Toon city, Ehime, 791-0295, Japan ; These authors contributed equally to this work.
Biomed Opt Express. 2014 Aug 29;5(10):3311-25. doi: 10.1364/BOE.5.003311. eCollection 2014 Oct 1.
Digital-scanned light-sheet microscopy (DSLM) illuminates a sample in a plane and captures single-photon-excitation fluorescence images with a camera from a direction perpendicular to the light sheet. This method is potentially useful for observing biological specimens, because image acquisition is relatively fast, resulting in reduction of phototoxicity. However, DSLM cannot be effectively applied to high-scattering materials due to the image blur resulting from thickening of the light sheet by scattered photons. However, two-photon-excitation DSLM (2p-DSLM) enables collection of high-contrast image with near infrared (NIR) excitation. In conventional 2p-DSLM, the minimal excitation volume for two-photon excitation restricts the field of view. In this study, we achieved wide-field 2p-DSLM by using a high-pulse energy fiber laser, and then used this technique to perform intravital imaging of a small model fish species, medaka (Oryzias latipes). Wide fields of view (>700 μm) were achieved by using a low-numerical aperture (NA) objective lens and high-peak energy NIR excitation at 1040 nm. We also performed high-speed imaging at near-video rate and successfully captured the heartbeat movements of a living medaka fish at 20 frames/sec.
数字扫描光片显微镜（DSLM）在一个平面上照亮样本，并使用相机从垂直于光片的方向捕获单光子激发荧光图像。这种方法对于观察生物标本可能是有用的，因为图像采集相对较快，从而减少了光毒性。然而，由于散射光子导致光片增厚而产生图像模糊，DSLM不能有效地应用于高散射材料。然而，双光子激发DSLM（2p-DSLM）能够通过近红外（NIR）激发收集高对比度图像。在传统的2p-DSLM中，双光子激发的最小激发体积限制了视野。在本研究中，我们通过使用高脉冲能量光纤激光器实现了宽视野2p-DSLM，然后使用该技术对小型模式鱼类青鳉（Oryzias latipes）进行活体成像。通过使用低数值孔径（NA）物镜和1040 nm的高峰值能量近红外激发，实现了宽视野（>700μm）。我们还以接近视频的速率进行了高速成像，并成功地以20帧/秒的速度捕获了活青鳉鱼的心跳运动。
