Mondia JP et al. (2011), Patterned femtosecond-laser ablation of Xenopus...

XB-ART-43674

Biomed Opt Express 2011 Aug 01;28:2383-91. doi: 10.1364/BOE.2.002383.

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Patterned femtosecond-laser ablation of Xenopus laevis melanocytes for studies of cell migration, wound repair, and developmental processes.

Mondia JP , Adams DS , Orendorff RD , Levin M , Omenetto FG .

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Ultrafast (femtosecond) lasers have become an important tool to investigate biological phenomena because of their ability to effect highly localized tissue removal in surgical applications. Here we describe programmable, microscale, femtosecond-laser ablation of melanocytes found on Xenopus laevis tadpoles, a technique that is applicable to biological studies in development, regeneration, and cancer research. We illustrate laser marking of individual melanocytes, and the drawing of patterns on melanocyte clusters to help track their migration and/or regeneration. We also demonstrate that this system can upgrade scratch tests, a technique used widely with cultured cells to study cell migration and wound healing, to the more realistic in vivo realm, by clearing a region of melanocytes and monitoring their return over time. In addition, we show how melanocyte ablation can be used for loss-of-function experiments by damaging neighboring tissue, using the example of abnormal tail regeneration following localized spinal cord damage. Since the size, shape, and depth of melanocytes vary as a function of tadpole age and melanocyte location (head or tail), an ablation threshold chart is given. Mechanisms of laser ablation are also discussed.

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Species referenced: Xenopus laevis
Genes referenced: hpse

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	Fig. 1. Schematic of femtosecond laser ablation of Xenopus tadpoles. (a) Femtosecond pulses were focused onto the specimens mounted on top of the motorized stage. For laser ablation, Xenopus laevis younger than stage 40 were held between a Petri dish with a glass welled bottom and a cover slip, while older tadpoles were placed inside a depression made of agar, secured with a glass cover slip, and then inverted for placement on the stage. (HWPâhalf wave plate, PBCâpolarizing beam cube, SPFâshort pass filter, DMâdichroic mirror, WLâwhite light source) (b) Images of Xenopus tadpoles at three different developmental stages.
	Fig. 2. (a) Absorption spectrum from a paste made of Xenopus tails and diluted in deionized water. Arrow indicates peak likely due to hemoglobin. The inset plots the absorption spectrum of melanin and oxygenated hemoglobin and the dashed line indicates the laser wavelength. (b) Histology section of undamaged tadpole tail (24 hpa) showing location of the notochord (N), the melanocytes surrounding the spinal cord (SC) and the dorsal muscle (DM). (c) Another section of the same tail where the red arrow points to absence of a melanocyte after laser treatment for one insult, i.e., shutter duration of 200ms and laser fluence of 26 mJ/cm2. (d) Section showing damage to SC and DM after multiple laser insults to melanocytes.
	Fig. 3. Different methods to mark melanocytes. (a) Top image is a melanocyte located on anterior dorsal side of a stage 46 Xenopus before laser ablation and the bottom image shows multiple ablated spots after a laser fluence of 2 mJ/cm2 and a shutter duration of 10 ms. (b) Tagging of individual melanocytes located near the tadpoleâs eye with triangles using a fluence of 2 mJ/cm2 and scan speed of 50 Âµm/s. (c) Before and after images of lines drawn on a dark wild-type stage 36 Xenopus for a 14 mJ/cm2 fluence and 150 Âµm/s scan speed. The red marker points to a long-lasting cavitation bubble. (d) Top image of melanocyte located on dorsal fin before laser ablation, middle image is the melanocyte after laser irradiation at 26 mJ/cm2 and shutter duration of 200 ms, and the bottom image shows a dark blue coloring of the ablated area, after bathing the Xenopus in the vital stain trypan blue, which indicates cell death.
	Fig. 4. Images of melanocyte migration after clearing a large area of melanocytes using laser ablation (raster scan with line spacing of 5 Î¼m, F = 6 mJ/cm2, scan speed = 150 Î¼m/s). Red arrowheads indicate the position of ablation (a), 19 hrs later (b), and 28 hrs later (c). (d) shows a dorsal image comparing the left (nonablated) and right (ablated) side of the Xenopus 28 hrs after laser ablation.
	Fig. 5. Tail regeneration after targeting the spinal cord. (a) Image of the position where the tail was amputated from a stage 40 Xenopus tadpole. The blue box highlights the region targeted by the laser. (b) Image after targeting laser in 10 locations inside blue box with laser fluence of 26 mJ/cm2 and a 200 ms shutter duration. The dark melanocytes that were targeted are gone. (c) An image showing the same tail after 1 week. (d) The normal shape of a regenerated tail.

References [+] :

Adams, A new tool for tissue engineers: ions as regulators of morphogenesis during development and regeneration. 2008, Pubmed