Research ArticleHEALTH AND MEDICINE

Precise closure of single blood vessels via multiphoton absorption–based photothermolysis

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Science Advances  15 May 2019:
Vol. 5, no. 5, eaan9388
DOI: 10.1126/sciadv.aan9388
  • Fig. 1 Multiphoton absorption–based SSP versus conventional CSP for anti-vascular treatment within the skin.

    (A) Before exposure. (B) After conventional CSP, all vessels within the light irradiation volume are denatured (indicated by the black-colored blood vessels). (C) With SSP, the target vessel is denatured/closed because the tightly focused fs laser deposits the radiant energy in the target vessel (the focal point, indicated by the magenta arrow) through multiphoton absorption, a nonlinear optical phenomenon occurring only at the focal point with high enough light power density. Other vessels and surrounding tissues are not affected. The inset shows that the vessels above the target vessel are not altered, although the fs laser pulses pass through them. Comparing (B) with (C), SSP could also treat vessels located deeper, where CSP could not reach due to the limited penetration depth for visible light.

  • Fig. 2 Imaging blood vessel before and after closure.

    (A and C) RCM and STD images of the same blood vessel before and after closure. (B) Algorithm of STD. N is the frame number for calculation of STD (in this case, N = 10); Ixy(i) is the pixel (x,y) intensity in the ith frame; Uxy is the average intensity at pixel (x,y) over all frames. (D to G) STD of different sized blood vessels before (top row) and after (bottom row) closure. The dashed white square boxes indicate the scanning irradiation area of the high-power treatment fs laser on the vessels. The pulse energy is 4.5 nJ per pulse, and the irradiation time is in the range of 0.1 to 2.1 s. Scale bars, 50 μm. (See movies S1 and S2 for more details.)

  • Fig. 3 Spatially selective blood vessel closure.

    (A and B) STD image of two blood vessels crossing over but located in different layers before (A) and after (B) treatment. The vertical blood vessel (white arrow) is at the bottom layer, and the other one (red arrow) is on the top layer. (C) Orientation of the two blood vessels. (D) RCM of the treated blood vessel at the bottom layer. The bright spot (white arrow) in (D) indicates the coagulation of blood cells. The laser pulse energy is 4.5 nJ per pulse, and the irradiation time is 4.5 s. Scale bars, 50 μm. (See movies S3 and S4 for more details.)

  • Fig. 4 Raman spectra of blood cells before (green curve) and after (red curve) the blood vessel closure.

    The Raman bands at 377 cm−1 (highlighted in vertical red line) and 970 and 1248 cm−1 (highlighted in magenta lines) increased, and the bands at 418 cm−1 (highlighted in dark green line) and 1543 cm−1 (highlighted in blue line) decreased. The bands at 677 and 754 cm−1 (black dashed lines) shifted to 667 and 749 cm−1, respectively. The red dashed lines in the images indicate the blood vessel inner edges. The white square represents the fs laser treatment area. The yellow dashed circle indicates the coagulation region. Scale bars, 50 μm. a.u., arbitrary units.

  • Fig. 5 Morphological dynamics of a large size blood vessel before, during, and after treatment using the point treatment system.

    (A to E) RCM images of the blood vessel before treatment, laser on, 1 s later, 2 s later, and after occlusion. The red dashed lines in (A) to (D) show the blood vessel edges. The red dot in (A) indicates the fs laser irradiation position. There is a bright spot [indicated by the arrow in (B)] at the fs laser irradiation position immediately after the laser is turned on. The bright spot becomes a bright stripe [as outlined by the white dashed curve in (C)] after the fs laser has irradiated for 1 s. A very bright spot appears after the fs laser has irradiated for 2 s, and the blood vessel begins to contract [as indicated by the arrow in (D)]. Scale bars, 50 μm (A to E). (F) Averaged intensity over an area surrounding the fs laser irradiation point (50 μm × 50 μm) variation with time. Time points (A) to (E) correspond to the images in panels (A) to (E), respectively. (See movie S6 for more details.)

  • Fig. 6 Partial blood vessel occlusion.

    (A and C) RCM and STD images of a blood vessel before treatment. The blood cells marked with red arrows can be clearly identified. (B and D) RCM and STD images of the same blood vessel after treatment. Scale bars, 50 μm. The white arrow in (B) indicates the coagulation part, corresponding to the dark area in the STD image (D). The flowing cells indicated by red arrows in (B) suggest that the blood vessel was partially blocked. (E) Speed of red blood cells (RBCs) before and after treatment. The average speed of RBCs increased significantly, from 0.021 to 0.029 mm/s (P < 0.05, by paired t test; n = 8). (See movie S7 for more details.)

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaan9388/DC1

    Fig. S1. Optical system for imaging and closing mouse ear blood vessels.

    Fig. S2. In vivo confocal Raman spectra of blood cells, sebaceous gland, and collagen.

    Fig. S3. Schematic of the point treatment system.

    Table S1. Tentative assignment of Raman bands.

    Movie S1. Y-shaped blood vessels before closure of the vertical blood vessel.

    Movie S2. V-shaped blood vessel after closure of the vertical blood vessel.

    Movie S3. Three-dimensional orientation of two blood vessels before treatment.

    Movie S4. Three-dimensional orientation of two blood vessels after treatment.

    Movie S5. fsRCM of the blood vessel during the fs laser treatment.

    Movie S6. The dynamic process of the blood vessel closure using the point treatment system.

    Movie S7. Partial closure of blood vessel.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Optical system for imaging and closing mouse ear blood vessels.
    • Fig. S2. In vivo confocal Raman spectra of blood cells, sebaceous gland, and collagen.
    • Fig. S3. Schematic of the point treatment system.
    • Table S1. Tentative assignment of Raman bands.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Y-shaped blood vessels before closure of the vertical blood vessel.
    • Movie S2 (.avi format). V-shaped blood vessel after closure of the vertical blood vessel.
    • Movie S3 (.avi format). Three-dimensional orientation of two blood vessels before treatment.
    • Movie S4 (.avi format). Three-dimensional orientation of two blood vessels after treatment.
    • Movie S5 (.avi format). fsRCM of the blood vessel during the fs laser treatment.
    • Movie S6 (.avi format). The dynamic process of the blood vessel closure using the point treatment system.
    • Movie S7 (.avi format). Partial closure of blood vessel.

    Files in this Data Supplement:

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