What Was Observed? (Introduction)

• Researchers used ultrafast (femtosecond) lasers to precisely target and remove melanocytes (pigment cells) in Xenopus laevis tadpoles.
• This technique is applied to study cell migration, wound repair, and overall developmental processes.
• By marking and ablating individual cells, the method allows tracking of cell movement and regeneration over time.

What Are Femtosecond Lasers and Melanocytes? (Background)

• Femtosecond lasers emit extremely short pulses (around 10⁻¹⁵ seconds), enabling very precise tissue removal.
• Xenopus laevis tadpoles are a well-established model for studying vertebrate development, regeneration, and even cancer-like behavior.
• Melanocytes are cells that produce melanin—the pigment that colors skin—and they absorb the laser light strongly.
• This high absorption makes melanocytes ideal targets for controlled laser ablation.

Materials and Methods

• A Ti:sapphire femtosecond laser operating at approximately 810 nm was used with 120 fs pulse duration and an 80 MHz repetition rate.
• The average power was adjustable between 20 mW and 1 W using neutral density filters and a half-wave plate.
• A mechanical shutter with precise opening (0.4 ms) and closing (0.6 ms) times controlled the pulse exposure.
• The laser beam was focused through an inverted microscope using 10x or 20x objectives.
• Tadpoles were mounted on a motorized stage; imaging and time-lapse recording allowed monitoring of cell migration.
• Proper anesthesia (tricaine or BTS) was used to immobilize the tadpoles during the procedures.
• Different mounting techniques were applied: younger tadpoles were placed in a glass-bottom dish and older ones in agar depressions for stable imaging.
• The damage threshold was determined by varying laser fluence until changes (contraction, expansion, or discoloration) were observed in the melanocytes.

What Happened in the Experiments? (Results)

• Ablation of Melanin-Containing Cells:
  • Scanning the laser over transparent regions caused no damage, but targeting melanocytes produced visible effects.
  • The extent of damage depended on the laser fluence and the duration of exposure—ranging from slight tissue contraction to fragmentation and bubble formation.
  • The depth and pigmentation of melanocytes affected the damage threshold; surface cells required lower energy than those deeper in the tissue.
• Laser Marking and Patterning:
  • The laser spot (~2 µm) is much smaller than a typical melanocyte (10–50 µm), allowing precise marking.
  • Different geometric patterns (spots, triangles, lines, grids, spirals) were drawn on individual cells or clusters to track their migration.
  • This method upgrades traditional in vitro scratch tests by performing similar experiments in living tissue.
  • Proper control of laser dosage prevented unwanted collateral damage like cavitation bubbles.
• Creating Collateral Damage for Functional Studies:
  • Laser ablation was also used to target melanocytes adjacent to the spinal cord, inducing localized spinal damage.
  • This targeted damage led to abnormal tail regeneration, showing that even small changes can affect developmental outcomes.
  • Variations in the position and extent of damage produced different tail shapes, demonstrating the sensitivity of regeneration to precise injuries.

Mechanisms and Key Conclusions (Discussion & Conclusion)

• Mechanisms of Laser Ablation:
  • At lower fluences, the process is driven by free-electron-induced chemical bond breaking in biomolecules.
  • At higher fluences, thermal effects accumulate, resulting in cavitation bubbles and more extensive tissue damage.
  • The melanin concentration in cells influences the absorption and overall efficiency of the ablation.
• Key Conclusions:
  • Femtosecond laser ablation is an effective tool for precisely marking, patterning, and ablating melanocytes in Xenopus tadpoles.
  • This method is valuable for in vivo studies of cell migration, wound healing, and regeneration.
  • The technique can be adapted for in vivo scratch tests and for loss-of-function experiments by selectively damaging tissues such as the spinal cord.
  • Overall, ultrafast laser techniques offer new insights into developmental biology and regenerative medicine.

观察到了什么？ (引言)

• 研究人员使用超快（飞秒）激光精确定位并去除非洲爪蟾蝌蚪中的黑色素细胞（含黑色素细胞）。
• 这种技术用于研究细胞迁移、伤口修复和整体发育过程。
• 通过标记和消融单个细胞，能够观察细胞如何移动和再生。

什么是飞秒激光和黑色素细胞？ (背景)

• 飞秒激光发出极短的脉冲（约10⁻¹⁵秒），能非常精确地去除组织。
• 非洲爪蟾蝌蚪是研究脊椎动物发育、再生和癌症样行为的成熟模型。
• 黑色素细胞产生黑色素，这种色素决定皮肤颜色，并且对激光波长有很高的吸收性。
• 这种高吸收性使黑色素细胞成为受控激光消融的理想目标。

材料和方法

• 使用钛宝石飞秒激光器，工作波长约810 nm，脉冲持续时间为120飞秒，重复频率为80 MHz。
• 通过中性密度滤光片和半波片调节平均功率，范围在20 mW至1 W之间。
• 机械快门精确控制脉冲曝光时间（开启0.4毫秒，关闭0.6毫秒）。
• 激光通过倒置显微镜和10倍或20倍物镜进行聚焦。
• 蝌蚪固定在电动平台上，使用CCD相机和延时摄影记录细胞迁移情况。
• 使用三氯乙醚或BTS对蝌蚪进行麻醉，以确保在激光手术过程中保持静止。
• 较年轻的蝌蚪放置于玻璃底培养皿中，而较年长的蝌蚪则置于琼脂凹槽中以保证成像稳定。
• 通过逐步增加激光能量直至观察到黑色素细胞变化来确定消融的损伤阈值。

实验中发生了什么？ (结果)

• 含黑色素细胞的消融：
  • 在透明区域扫描激光不会造成损伤，但定向照射黑色素细胞会产生明显效果。
  • 损伤程度取决于激光能量和脉冲持续时间，可能表现为组织收缩、细胞碎裂或气泡形成。
  • 黑色素细胞的深度和色素含量影响损伤阈值；表层黑色素细胞所需能量较低，而较深的细胞需要更高能量。
• 激光标记和图案绘制：
  • 激光光斑约2微米，远小于典型黑色素细胞（10–50微米），因此可以进行精细标记。
  • 成功在单个细胞或细胞群上绘制了点、三角形、线条、网格和螺旋等多种几何图案。
  • 这些图案有助于追踪细胞迁移和再生，类似于体外划痕实验，但在活体内进行。
  • 精确控制激光剂量可避免因气泡形成而引起的非目标组织损伤。
• 利用黑色素细胞制造附带损伤进行功能缺失研究：
  • 激光消融也用于定向照射靠近脊髓的黑色素细胞，从而间接损伤脊髓。
  • 这种局部损伤导致尾巴再生异常，证明即使是微小的损伤也会影响发育结果。
  • 不同位置和不同程度的脊髓损伤会导致再生尾巴形态出现明显变化。

机制及主要结论 (讨论与结论)

• 激光消融机制：
  • 低能量时，消融主要由自由电子引起的化学键断裂驱动。
  • 高能量时，热效应累积导致气泡形成和更大范围的组织损伤。
  • 细胞内黑色素的浓度会影响吸收效率，从而决定消融效果。
• 主要结论：
  • 飞秒激光消融技术能精确标记、绘制图案并去除蝌蚪中的黑色素细胞。
  • 该方法适用于研究活体中细胞迁移、伤口修复和再生过程。
  • 激光消融技术可以用于体内划痕实验和功能缺失研究，揭示组织修复及发育信号传导机制。
  • 该技术为研究癌症样细胞行为和局部脊髓损伤对尾巴再生的影响提供了新的、可控的实验手段。
  • 总体来看，超快激光技术在发育生物学和再生医学研究中具有广阔的应用前景。