Current Optics and Photonics

  • 제2권6호
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  • Pages.595-605
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  • 2018
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  • 2508-7266(pISSN)
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  • 2508-7274(eISSN)
한국광학회 (Optical Society of Korea)
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High-speed Two-photon Laser Scanning Microscopy Imaging of in vivo Blood Cells in Rapid Circulation at Velocities of Up to 1.2 Millimeters per Second

  • Boutilier, Richard M. (School of Mechanical Engineering, Kyungpook National University) ;
  • Park, Jae Sung (School of Mechanical Engineering, Kyungpook National University) ;
  • Lee, Ho (School of Mechanical Engineering, Kyungpook National University)
  • 투고 : 2018.08.23
  • 심사 : 2018.10.02
  • 발행 : 2018.12.25
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초록

The two-photon process of microscopy provides good spatial resolution and optical sectioning ability when observing quasi-static endogenous fluorescent tissue within an in vivo animal model skin. In order to extend the use of such systems, we developed a two-photon laser scanning microscopy system capable of also capturing $512{\times}512$ pixel images at 90 frames per second. This was made possible by incorporating a 72 facet polygon mirror which was mounted on a 55 kRPM motor to enhance the fast-scan axis speed in the horizontal direction. Using the enhanced temporal resolution of our high-speed two-photon laser scanning microscope, we show that rapid processes, such as fluorescently labeled erythrocytes moving in mouse blood flow at up to 1.2 mm/s, can be achieved.

키워드

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FIG. 1. Schematic diagram of the high-speed two-photon and confocal laser scanning microscope.

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FIG. 2. Timing diagram of the H-sync and V-sync control signals and the analog angular position voltage signal to the galvanometer.

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FIG. 3. Representative images of auto-fluorescent structures found in auricle skin and scanned at an imaging rate of 180 FPS, and averaged over 30 frames, for subsequent layers in ex vivo mouse tissue. All images are 512 × 220 pixels (220 μm × 95 μm FOV).

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FIG. 4. Stationary ex vivo DiI-labeled erythrocytes translated at a fixed speed of 1200 μm/s via a motorized sample stage.

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FIG. 5. Sample image of in vivo low blood flow captured at 180 FPS. (a) A single frame composite of the confocal channel, shown in red, and the two-photon DiI selection channel shown in blue. (b) The same single frame after being despeckled and isolated to the confocal channel. Individual cells are visible within the drawn-in outline of the blood vessel. (c) The same single frame but isolated to the DiI selection channel. A single labeled erythrocyte is visible within the drawn-in outline of the blood vessel as well as a weakly overlapping auto-fluorescent signal from the collagen in the blood vessel’s wall.

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FIG. 6. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~130 μm/s through a blood vessel. The vessel’s walls are marked in red outline.

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FIG. 8. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~1200 μm/s through a blood vessel. The vessel’s walls are marked in red outline.

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FIG. 7. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~600 μm/s through a blood vessel. The vessel’s walls are marked in red outline.

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