Selective disruption of vascular endothelium of zebrafish embryos by ultrafast laser microsurgical treatment Suk-Yi Woo,1 Heh-Young Moon,1,2 Tag Gyum Kim,1 Heung Soon Lee,1 Mehra S. Sidhu,1 Changho Kim,1,3 Jae-Phil Jeon,3 and Sae Chae Jeoung1,* 1

Division of Advanced Technology, Korea Research Institute of Standards and Science, 1-Doryong-Dong, YuseongGu, Daejeon 305-340, South Korea 2 Department of Nano Chemistry, Gacheon University, Seongnam, Gyeonggi-do 461-701, South Korea 3 L2K Co., Ltd.,Yuseong-Gu, Daejeon 305-500, South Korea * [email protected]

Abstract: In this work, we demonstrate that ultrafast laser irradiation could selectively disrupt vascular endothelium of zebrafish embryos in vivo. Ultrafast lasers minimize the collateral damage in the vicinity of the laser focus and eventually reduce coagulation in the tissues. We have also found that the threshold fluence for lesion formation of the vascular endothelium strongly depends on the developmental stage of the embryos. The threshold laser fluence required to induce apparent lesions in the vascular structure for Somite 14, 20 and 25 stages is about 5 J/cm2 ~7 J/cm2, which is much lower than that for the later development stages of Prim 16 and Prim 20 of 30 J/cm2 ~50 J/cm2. The proposed method for treating the vascular cord of zebrafish embryos in the early stage of development has potential as a selective and effective method to induce a fatal lesion in the vascular endothelium without damaging the developed blood vessels. ©2015 Optical Society of America OCIS codes: (320.7130) Ultrafast processes in condensed matter, including semiconductors; (140.0140) Lasers and laser optics; (170.3890) Medical optics instrumentation.

References and links 1.

S. Isogai, N. D. Lawson, S. Torrealday, M. Horiguchi, and B. M. Weinstein, “Angiogenic network formation in the developing vertebrate trunk,” Development 130(21), 5281–5290 (2003). 2. N. D. Lawson and B. M. Weinstein, “In vivo imaging of embryonic vascular development using transgenic zebrafish,” Dev. Biol. 248(2), 307–318 (2002). 3. W. J. Li and N. Xi, “Novel micro gripping, probing, and sensing devices for single cell surgery,” in Proceedings of the IEEE Conference on Engineering in Medicine and Biology Society (IEEE, 2004), pp. 2591–2594. 4. T. Juhasz, F. H. Loesel, R. M. Kurtz, C. Horvath, J. F. Bille, and G. Mourou, “Corneal refractive surgery with femtosecond lasers,” IEEE J. Sel. Top. Quantum Electron. 5(4), 902–910 (1999). 5. N. Shen, C. B. Schaffer, D. Datta, and E. Mazur, “Photodisruption in biological tissues and single cells using femtosecond laser pulses,” in Proceeding of IEEE Conference on Lasers and Electro-Optics (IEEE, 2001), pp. 403–404. 6. A. Vogel and V. Venugopalan, “Mechanisms of pulsed laser ablation of biological tissues,” Chem. Rev. 103(2), 577–644 (2003). 7. N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P. D. Lyden, and D. Kleinfeld, “Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke,” Nat. Methods 3(2), 99– 108 (2006). 8. S. C. Jeoung, M. S. Sidhu, J. S. Yahng, H. J. Shin, and G. Y. Baik, “Application of ultrafast laser optoperforation for plant pollen walls and endothelial cell membranes,” in Advances in Lasers and Electro Optics (Intech, 2010). 9. U. K. Tirlapur and K. König, “Targeted transfection by femtosecond laser,” Nature 418(6895), 290–291 (2002). 10. K. Oikawa, S. Matsunaga, S. Mano, M. Kondo, K. Yamada, M. Hayashi, T. Kagawa, A. Kadota, W. Sakamoto, S. Higashi, M. Watanabe, T. Mitsui, A. Shigemasa, T. Iino, Y. Hosokawa, and M. Nishimura, “Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis,” Nature Plants 1(4), 15035 (2015).

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4694

11. B. Rethfeld, K. Sokolowski-Tinten, D. von der Linde, and S. I. Anisimov, “Ultrafast thermal melting of laserexcited solids by homogeneous nucleation,” Phys. Rev. B 65(9), 092103 (2002). 12. C.-T. Yang, R. D. Sengelmann, and S. L. Johnson, “Larval melanocyte regeneration following laser ablation in zebrafish,” J. Invest. Dermatol. 123(5), 924–929 (2004). 13. M. Yamaguchi, E. Yoshimoto, and S. Kondo, “Pattern regulation in the stripe of zebrafish suggests an underlying dynamic and autonomous mechanism,” Proc. Natl. Acad. Sci. U.S.A. 104(12), 4790–4793 (2007). 14. J. E. Jones and J. T. Corwin, “Regeneration of sensory cells after laser ablation in the lateral line system: hair cell lineage and macrophage behavior revealed by time-lapse video microscopy,” J. Neurosci. 16(2), 649–662 (1996). 15. T. Roeser and H. Baier, “Visuomotor behaviors in larval zebrafish after GFP-guided laser ablation of the optic tectum,” J. Neurosci. 23(9), 3726–3734 (2003). 16. M. F. Yanik, H. Cinar, H. N. Cinar, A. D. Chisholm, Y. Jin, and A. Ben-Yakar, “Neurosurgery: Functional regeneration after laser axotomy,” Nature 432(7019), 822 (2004). 17. V. Kohli and A. Y. Elezzabi, “Laser surgery of zebrafish (Danio rerio) embryos using femtosecond laser pulses: Optimal parameters for exogenous material delivery, and the laser’s effect on short- and long-term development,” BMC Biotechnol. 8(1), 7 (2008). 18. M. S. Sidhu, M. Y. Choi, S. Y. Woo, H. K. Lee, H. S. Lee, K. J. Kim, S. C. Jeoung, J. S. Choi, C. K. Joo, and I. H. Park, “Femtosecond laser-assisted selective reduction of neovascularization in rat cornea,” Lasers Med. Sci. 29(4), 1417–1427 (2014). 19. C. B. Kimmel, W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling, “Stages of embryonic development of the zebrafish,” Dev. Dyn. 203(3), 253–310 (1995). 20. B. Fouquet, B. M. Weinstein, F. C. Serluca, and M. C. Fishman, “Vessel patterning in the embryo of the zebrafish: guidance by notochord,” Dev. Biol. 183(1), 37–48 (1997). 21. S. Childs, J. N. Chen, D. M. Garrity, and M. C. Fishman, “Patterning of angiogenesis in the zebrafish embryo,” Development 129(4), 973–982 (2002). 22. B. C. Stuart, M. D. Feit, S. Herman, A. M. Rubenchik, B. W. Shore, and M. D. Perry, “Optical ablation by highpower short-pulse lasers,” J. Opt. Soc. Am. B 13(2), 459–467 (1996). 23. A. P. Joglekar, H.-H. Liu, E. Meyhöfer, G. Mourou, and A. J. Hunt, “Optics at critical intensity: Applications to nanomorphing,” Proc. Natl. Acad. Sci. U.S.A. 101(16), 5856–5861 (2004). 24. H. Y. Moon, M. S. Sidhu, H. S. Lee, and S. C. Jeoung, “Dynamic changes in PDMS surface morphology in femtosecond laser treatment,” Opt. Express 23(15), 19854–19862 (2015).

1. Introduction The genetic accessibility and optical clarity of developing zebrafish embryos make them a useful model system to examine the mechanisms of blood vessel formation and patterning during development [1]. Transgenic zebrafish that express green fluorescent protein (GFP) throughout vasculature is also a valuable model for investigating the dynamics of vessel formation in vascular endothelium under the control of zebrafish fli1 promoters though enabling in vivo time lapse imaging of the vascular endothelial cells in developing and living embryos [2]. Ultrafast laser pulses are known to be useful in many biological applications such as micro-manipulation and dissection of nano-scale and micro-scale structures in live cells and other biological materials [3–10]. There are two main advantages in using femtosecond Ti:Sapphire laser outputs for the tiny ablation of live samples. First, the laser wavelength at near-infrared region is able to provide a large penetration depth in tissues. Second, with pulse durations of approximately 100 fs, low energy per pulse is necessary for the surgery [4, 5] because the threshold laser fluence to induce alterations in the targets decreases significantly when using a shortened laser pulse width. Meanwhile, long pulsed laser microsurgery requires two to three orders of magnitude higher energy [11]. The low energy could minimize collateral damage in the vicinity of the laser focus and reduce the possibility that the cell be damaged. Several reports have also documented the application of lasers for the micro-treatment of zebrafish. The laser systems used for the ablation of larval pigments cells [12], melanocytes [13], sensory cells [14], and optic tectums [15] include Q-switched Nd:YAG lasers and nitrogen lasers with pulse durations of several nanoseconds [12–15]. Yanik et al. showed that ultrafast lasers have the potential to perform precise axotomy in a simple organism (Caenorhabditis elegans), which has only 302 neurons, while observing the functional regeneration of the operated axons following the surgery [16]. Kohli et al. also reported that

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4695

fluorescein isothiocyanate (FITC) was successfully introduced into blastomere cells of zebrafish embryos without significant differences in hatching rates and developmental morphologies [17]. We have also investigated fs-laser-based microsurgery for selective treatment of rat corneal neovascularizations (in vivo) [18]. We reported that the histological and OCT (Optical Coherent Tomography) characterization of the anterior segments within the corneal stoma exhibited localized degeneration of the neovascular structures without harmful effects in adjacent tissues of rat cornea. In this work, we have applied fs-laser pulses to selectively induce damage on vascularrelated structures in zebrafish embryos at different developmental stages. We have found that the threshold fluence for lesion formation of the vascular endothelium strongly depends on the developmental stage of the embryos. The results reveal that ultrafast laser microsurgery can be successfully utilized for selective and effective removal of neovascular structures in the early developmental stages of zebrafish embryos. Within our knowledge, this work is the first application of ultrafast laser on micro-surgery of vascular endothelium of Zebrafish embryos. 2. Materials and methods 2.1. Zebrafish Transgenic zebrafishs (TG (fli1: EGFP) y1 line) are commercially available through the zebrafish international resource center (Eugene, OR; http://www.zebrafish.org/home/guide.php). The zebrafish incubation temperature of the water was maintained at 28.5 ± 1 °C and the incubators were on a 10 hr dark/14 hr light cycle. Adult male and female TG (flil: EGFP)y1 zebrafish were placed in a breeding tank containing 1.5 L water. In each breeding tank, one adult female was paired with two adult males. A total of 2 tanks were set up for simultaneous breeding. The embryos were harvested at approximately 40 min after the start of the light cycle. A 2 mm wire mesh was placed at the bottom of each breeding tank to protect the fallen eggs from being eaten by the adult fish. Five different developmental stages for the embryos were examined in the current work. We have followed the description of embryos developmental stages appeared in the previous publication by C. B. Kimmel et al. in naming of the stages in zebrafish embryos [19]. The phenotype of the embryos was utilized to differentiate the embryo developmental stages including 14, 20, and 25 somite, and Prim 16 and 20. The embryo’s vascular structure began to form in the 14 somite stage. While the network of blood vessels was not fully developed yet, the beginning of the lumen formation was evident in the dorsal aorta in the 24 somite stage. The first intersomitic arteries sprouted from the dorsal aorta in the 26 somite stage. In the further development of the embryos to the Prim stage, the vascular cord had an apparent lumen with high density endothelial cells at the blood vessel wall [20, 21]. 2.2. Optical setup for microsurgery Figure 1 presents the schematic diagram for the ultrafast laser photodisruption setup for zebrafish embryo’s vascular structure. A home-made confocal laser scanning microscope was used to visualize and monitor the vascular structure of the transgenic zebrafish embryos in vivo. An argon ion laser (Coherent, Innova 90, USA) at the wavelength of 488 nm is focused into the zebrafish embryos through an objective lens with a numerical aperture (N.A.) of 0.5. Scanning of the laser beams in the lateral plane was accomplished by using a galvano-scanner (Cambridge Technology Inc. 6230H, USA). A pinhole with a diameter of 50 μm was inserted before the scanner. The laser-induced fluorescence from GFP in the endothelial cell of the transgenic zebrafish embryos was optically filtered out from the scattered laser light using a dichroic mirror and it was further filtered out with a narrow optical band pass filter with a center wavelength for the GFP. The emitted photons were

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4696

detected by using a photomultiplier tube (PMT, Hamamatsu, R928, Japan) and fed into a personal computer for further data processing.

Fig. 1. Ultrafast laser microsurgery setup for the vascular structure and blood vessel of transgenic zebrafish. The fundamental output from the amplified ultrafast laser was delivered to the vascular structure through an objective lens (N.A. = 0.5). The fluorescence images were simultaneously measured by scanning an Argon ion laser through the same objective lens. The laser power of the argon-ion laser was kept low in order not to induce changes in the in situ fluorescence images.

The ultrafast laser microsurgery system is based on an amplified Ti:Sapphire laser system (Quantronix, Integra, USA) operating at a wavelength of 800 nm, a pulse duration of 150 fs, and a pulse repetition rate of 1 kHz. The laser fluence was controlled by using a variable neutral density filter (Sigma Koki, VND, Japan). All the experiments were conducted under a single-pulse configuration utilizing two mechanical shutters (Uniblitz, LS672, USA) with an opening time constant less than 0.5 ms. One of the two mechanical shutters is normally operated in opening position and closed upon external triggering, of which signal is fired by manual switch and synchronized with a high voltage signal from Pockels cell. Meanwhile the other one is normally closed and opened upon the above triggering signal with a time delay. We have confirmed only single pulses by monitoring photodiode signals with an oscilloscope (Tektronix, TDS5104B, USA). For the photo-induced disruption and characterization of the zebrafish embryo vascular structure, we first placed the zebrafish embryos in the focal plane of the microscopy and observed the confocal fluorescence images of the embryo vascular structure. We manipulated the XY-stage of the microscope in order to place the vascular endothelium in the focal point of the ultrafast laser beam and irradiated single laser pulse into the target vascular structure of the embryos. The fluorescence images were obtained during and after the laser treatment. After the ultrafast laser treatment, the embryos were bred in a fish bowl in order to determine whether the laser-treated embryos could recover and successfully hatched. Some laser-treated embryos were fixed for further histological evaluation. In the current study, the embryos were not treated with anesthetic. 2.3. Histological analyses The location of each laser-exposed site was carefully mapped in relation to the vascular endothelial pattern of the embryos. The zebrafish embryos were fixed overnight in 4%

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4697

paraformaldehyde after microsurgery and then immersed in PBS (Phosphate Buffered Saline) containing 30% sucrose at room temperature for 2 hrs for osmosis in the fixed tissue. They were embedded in tissue freezing medium (Leica, Germany) and later transferred to refrigerator at –20 °C for 4-6 hrs. The frozen embryos were cryo-sectioned with a thickness of 20 μm (Leica CM1850 cryotome) and stained in hematoxylin and eosin (H&E). Brightfield photographs of the slices were obtained using an optical microscopy (Axioscope 2, Zeiss, Germany). The H&E stained sections were also imaged using a high-resolution optical microscope. 3. Results Figure 2 presents a series of confocal fluorescence images of the vascular structure in four different developmental stages of transgenic zebrafish embryos before and after the irradiation of the ultrafast laser beam into the targeted area (indicated by arrows). While the vascular structure in the 16 somite stage of the embryos was completely disrupted after the ultrafast laser irradiation, which was grouped into ‘complete disruption’. At the development stage of 20 somite and 25 somite, the vascular cord operated by ultrafast laser was reconnected with a change in its shape, which is grouped into ‘half recovery’. The blood vessels at a more developed stage of the embryos, e.g. the Prim 16 stage, were fully recovered within a second, which was grouped into ‘perfect recovery’.

Fig. 2. Typical confocal microscopic images (1) before and (2 and 3) after the ultrafast photoinduced disruption of the vascular structure in zebrafish embryos in four different developmental stages. The images were taken with a time lapse of 0.5 sec. The black arrows indicate the targeted area of the ultrafast laser beam. Scale bar: 20 μm.

The location of each laser-irradiated site of the vascular structure was examined through sequential sectioning of the laser treated embryos with a thickness of 20 μm. Figure 3 presents the optical images of the sectioned slices, which were obtained through successive sectioning and histological analyses of the embryos in 16 somite stage treated with laser irradiation with a fluence of 7.4 J/cm2. The sectioned slices were stained using H&E. The sequential images from Fig. 3(a) to Fig. 3(f) were taken from the slices of the embryos which

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4698

were successively sectioned. As shown in Fig. 3(a), the outer border line of the section with a color of strong violet indicates the nucleus of vascular endothelial cell while the area with red colors inside the sections presents cytosol. Figure 3(b) and Fig. 3(e)-3(f) also exhibit intact vascular structure at the border line of the sections with slightly extruded yolk cells from the embryos. As shown in Figs. 3(c) and 3(d), however, the lesions of the apparent disruption of vascular endothelial cell inside yellow circles were observed with a spout-like shape of extrude yolk. It should be noted that the lesions were limited only to the vascular structure of the embryos. Only the vascular structure layer with the superficial nuclei was removed. It should be also noted that there was no evidence of coagulation in the photo-disruptive lesions. Since the thickness of the sections is 20 μm, we can conclude that the lateral damage size upon ultrafast laser exposure at a laser fluence of 7.4 J/cm2 should be limited to 40 μm as an upper limit from the histological analysis. With decreasing the laser fluence, the singlepulse laser irradiation induced damage in the vascular structure with a significantly smaller size. The results from the current histological analyses can be considered by direct evidence for the realization of photodisruption of the vascular structure of zebrafish embryos by utilizing ultrafast laser irradiation with high precision. Furthermore, the vascular structure of the embryos can be effectively disrupted without altering other areas.

Fig. 3. Series of optical images of the zebrafish embryo sections. The images from (a) to (f) were successively obtained from the samples sectioned by crytome with a thickness of 20 μm. Only the middle sections (c-d) exhibit complete disruption marked with yellow circles of the angiogenesis in addition to the feature of yolk cell extrusion. However, Figs. 3(a)-3(b) and 3(e)-3(f) reveal only the extruded yolk cells from the embryos and they maintain intact vascular structure parts at the border. Scale bar: 100 μm.

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4699

Fig. 4. The lowest laser fluence to induce a disruption of the zebrafish embryo’s vascular structure for different development stage: the values of the threshold fluence were determined via monitoring the fluorescence images of the vascular cord before and after the laser irradiation as depicted in Fig. 2. All experiments were conducted using a single shot configuration.

By observing the confocal fluorescence images before and after the laser irradiation with changing the laser fluence, we have determined the lowest laser fluence that induces an apparent change in the shape of the vascular structure of the zebrafish embryos. Furthermore, we have statistically determined the threshold fluence for the lesion in vascular structures of zebrafish embryos. In order to determine the threshold fluence, more than 100 embryos were examined in triplicate for each developmental stage. In total, 431 embryos were examined with varying the laser fluence. Figure 4 illustrates the threshold laser fluence required to induce laser lesions in the vascular structure with different development stages of the embryos. All the experiments were conducted under a single pulse configuration. The laser fluence of approximately 5 J/cm2 ~7 J/cm2 was sufficiently high to induce the apparent disruption of the vascular structure for Somite 14, 20 and 25 stages. However, the laser fluence level of 30 J/cm2 ~50 J/cm2 create apparent lesions of the blood vessels in the later development stages of Prim 16 and Prim 20. Table 1. Number of zebrafish embryos for lesion typesa

14 somites 20 somites

Complete disruption T D 32 10 26 3

Half recovery T 29 37

Perfect recovery D 3 7

T 19 36 17

D 2 2 2

25 somites 9 0 13 0 Prim 16 4 1 2 29 0 Prim 20 1 0 1 0 24 0 a The numbers of total treated embryos (T) and dead embryos (D) for each group of lesion types are denoted as a function of the embryo’s development stages.

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4700

Fig. 5. The percent probabilities for each lesion type for complete disruption (red), half recovery (green), and perfect recovery (blue) with variations in the developmental stages of the embryos.

304 embryos in five different developmental stages were examined in order to know whether the recovery rate of the laser treated vascular structure and the fate of the embryos were dependent on the embryo’s developmental stage. All examined embryos after the fslaser irradiation with the laser fluence of 7.4 J/cm2 were tentatively grouped into three types of lesions: perfect recovery, half recovery, and complete disruption of the vascular structure, as described above. The laser fluence of 7.4 J/cm2 is sufficiently high enough to make the detrimental lesions on the vascular endothelial cell for the early stage of embryo’s development such as Somite 14, 20, and 25, while it is well below the photo-disruption threshold fluence for the late stage of embryo’s development such as Prime 20. The results are summarized in Table 1. The number of embryos for each group of lesion type is denoted by ‘T’ in Table 1. The correlation statistics of the different types of lesions in the vascular structure is presented in Fig. 5 as a function of the developmental state of the embryos. The probability for each group of lesions was strongly dependent on the status of the embryo development. In the early developmental stage of 14 somite, approximately 40% of the treated vascular structure was completely disrupted without recovery. However, with further development of the blood vessel structure, the lesions in the blood vessels in the Prim 20 stage recovered within several seconds with a high probability of more than 92%.

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4701

Fig. 6. The typical final status for each development stage of the laser-treated zebrafish embryos. (a) embryo did not progress further in development after the laser irradiation; (b) embryo developed but did not hatch and eventually died; (c) embryo hatched but the juvenile fish did not have a good shape; and (d) embryo finished its developmental process, hatched well, and grew normally even though the embryo’s vascular structure was treated with an ultrafast laser.

Figure 6 presents typical final status of the laser-treated zebrafish embryos. As shown in Fig. 6(a), some laser-treated embryos were not able to develop after the laser treatment and they eventually died. Figure 6(b) presents an embryo that further developed but did not hatch. The laser-treated embryo in Fig. 6(c) successfully developed and hatched, but the juvenile fish did not have a good shape. The embryo presented in Fig. 6(d) finished its development process, hatched well, and grew normally even though its vascular structure underwent the operation. The fate of the embryos after the laser treatment on the vascular endothelium was also strongly dependent on the embryos development stage. The number of dead embryos for each group of lesion types is denoted by ‘D’ in the Table 1. Most embryos in the perfect recovery group recovered and were successfully hatched. However, approximately 35% of the embryos in the 14 somite stage, of which the vascular structure was completely disrupted, did not develop further and eventually died. It is evident that the current method of treating the vascular structure based on ultrafast laser irradiation has potential to selectively and effectively induce a fatal lesion in the vascular structure without significant perturbation in a developed blood vessel. It is of great interesting to discuss about the underlying mechanism that governs the dependence of both the laser fluence threshold for the disruption of vascular structure and its statistical correlation of lesion types on the development status of the zebrafish embryos. The current work unequivocally reveals that the vascular endothelium in the early development stage is very vulnerable to the ultrafast laser irradiation compared with those in later developed states. Unless conclusive evidence is available in this stage, the physical dimension of the vascular structure should be an important factor in the photo-induced disruption of the vascular structure. If the wall thickness of the vascular structure in the early stage is significantly less than that in the later stage, the vascular structure in the early stage

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4702

should be significantly affected by the ultrafast laser irradiation with keeping the ablation depth caused by the single-pulse ultrafast laser constant. Even if the ablation depth is constant with variations in the nature of the vasculogenesis, the relative portion of damaged area in the early development stage of the embryos is apparently significant compared with that for further developed vascular structure. Another explanation might be the difference in either the physical characteristics, e.g. elastic constant, or the physiological properties, e.g. self-healing process, of the lesions of the vascular cord. The vascular cord of the embryos in the early development stage should be small in size and have a lower density of in endothelial cells compared with that in further developed embryos. At a cellular level, angiogenesis involves localized endothelial cell proliferation and migration, followed by remodeling of the nascent vessel, with the latter including the removal, growth, or subdivision of the new channel [20, 21]. The first intersomitic arteries sprout from the dorsal aorta in the 26 somite stage. At this time, the network of all major vessels is not fully open, although the beginning of the lumen formation is evident in the dorsal aorta in the 24 somite stage. With further development of the embryos to the Prim stage, the vascular cord has a lumen with high density endothelial cells in the blood vessel wall. It is necessary to systematically investigate the dependence of the lesion probability of the vascular structure with changing the vascular structure depending on the nature of the cord in specific development state of the embryos. Finally, it is of interesting to discuss about these fast recovery observed in Prim 16 stage of the embryos after laser surgery. When intense fs laser pulses are focused on the vascular endothelium of the zebrafish embryo, the density of the electron should increase due to tunneling and avalanche ionization accompanied with multiphoton ionization [22, 23]. The increase in electron density leads to an additional absorption of fs laser and the high-density plasma expands with hypersonic velocity that drives a shockwave in addition to the apparent ablation of the target materials. The shockwave plays a role in photoinduced damages of the vascular structures, which could eventually disrupt the vascular structures. Meanwhile, it should be noted that highly intense optical radiation should induce apparent photo-bleaching of GFP in the vascular endothelial cells and cause a depletion of fluorescence intensity at laser irradiated region even if the laser fluence is not high enough to disconnect the vascular structures without complete disruption of the vascular structure. This could induce an immediate recovery just after laser irradiation on vascular endothelium if the fluorescent protein could be recovered within one second. Considering that the size of damage shown in fluorescent images of several tens of micrometers is comparable to the size of single endothelium cell, it is not probable to migrate into the focal position within one second to recover the fluorescence intensity. The another possible explanation is a dynamical changes in the portion of vascular endothelium close to the laser irradiated position caused by the mechanical forces accompanied by the shockwaves. We have recently reported that the effect of the dynamics of crater size on the elastomer of poly(dimethylsiloxane) (PDMS) morphology in fs-laser micro-processing [24]. The crater formed on vascular endothelium should deform out of focal position for the confocal microscopic measurement after laser irradiation and be eventually restored to the steady state. Even if we have no any quantitative description for the time constant for the changes in fluorescent images upon irradiating the laser pulse, we can conjecture that the origin of the dynamics related on that time constant should be related on the modulus of elasticity for endothelium vascular structures. Without complete knowledge about the biomechanical properties related on those transient phenomena, no clear statement could be given on the time constant at this stage. It should be necessary to investigate the effect of the modulus of elasticity as well as applied shear forces on the dynamics of the damage size in more detailed.

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4703

7. Conclusion The current work revealed that micro-surgery based on ultrafast laser pulses has a potential to selectively disrupt a transgenic zebrafish embryo’s vascular cord. Using different developmental stages of embryos including 14, 20, and 25 somite, and Prim 16 and 20, we investigated the opto-disruption on the vascular structure of zebrafish embryos in vivo. Ultrafast lasers can be precisely focused on the structure of the embryos by utilizing confocal fluorescence microscopy, which can be used to visualize specifically the endothelial cells in the transgenic zebrafish embryos. Histological analyses on the sectioned slices of the operated embryos with a thickness of 20 μm revealed that the lateral dimension of the affected part under the current operation method was approximately 40 μm as an upper limit. The threshold fluence and statistical correlation of the photo-induced disruption probability of the vascular structure as well as the fate of the laser-treated embryos were strongly dependent on the development stages. Furthermore, the observations led us to suggest that ultrafast laser operations on blood vessels should be an effective method of intra-tissue surgery applications with minimized detrimental effects. Many studies have been already performed with ultrafast lasers under the fully developed blood vessels. Meanwhile, we have conducted the experiments with an early angiogenetic network. It is not easy to differentiate endothelial cell lines corresponding to neovascular structures at early developed stage from the periphery of the soft tissues. The key findings from this study are that the ultrafast laser removal of the vascular structure is much more effective on somitogenesis in early development stages compared with that under more developed stages. It is meaningful to investigate any specific idea for selective way to make detrimental lesion on neovascular structure. The current findings reveal the potential of a remedy for many different types of angiogenesis related diseases, e.g. typical retina defects that are caused by numerous types of pathogenesis. Acknowledgment This work was financially supported by the KRISS program (15011058).

#248408 Received 21 Aug 2015; revised 30 Oct 2015; accepted 2 Nov 2015; published 4 Nov 2015 (C) 2015 OSA 1 Dec 2015 | Vol. 6, No. 12 | DOI:10.1364/BOE.6.004694 | BIOMEDICAL OPTICS EXPRESS 4704

Selective disruption of vascular endothelium of zebrafish embryos by ultrafast laser microsurgical treatment.

In this work, we demonstrate that ultrafast laser irradiation could selectively disrupt vascular endothelium of zebrafish embryos in vivo. Ultrafast l...
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