New generation high-energy tunable femtosecond laser for nonlinear optical microscopes to investigate advanced imaging of ophthalmic tissue

近年来激光技术的创新在超短激光脉冲的产生和应用方面取得了显著的进步。飞秒激光现在被科学家用来“观察”一个分子发生化学反应时的分解过程，也被外科医生用来以前所未有的精度进行激光手术。超快激光技术仍在快速发展，最终短脉冲可能很快就会用于亚微米精度的激光眼科手术。我们的长期目标是提高我们对眼睛成像的能力，以提高加拿大人的眼睛健康质量。为了实现这一目标，一项技术挑战是能够清晰地成像飞秒激光产生的极小的“切口”。这需要以亚微米级的空间分辨率对眼睛内部结构进行实时三维成像。我们建议同时使用多种（多模态）非线性光学显微镜方法，以获得更全面的样品信息。一种重要的方法，被称为受感染相干反斯托克斯拉曼散射（e-CARS）显微镜，是实现这种终极成像的理想方法。e-CARS显微镜提供了一种独特的方法，通过使用分子振动作为对比机制来成像生物样品。e-CARS显微镜能够以亚微米分辨率对微小结构进行三维成像，并且可以很容易地实现到激光手术系统中，因为激光手术中使用的相同光学元件也可以用于e-CARS显微镜。因此，体内e-CARS显微镜将大大有助于提高飞秒激光眼科手术的精度。另一个潜在的重要应用是年龄相关性黄斑变性（AMD）的诊断和随访。这种视网膜疾病是加拿大法定失明的主要原因。大约25%的75岁以上的加拿大人在不同程度上受到影响。e-CARS显微技术的发展和应用将有助于更好地了解这种疾病的病理生理，并有助于找到治疗AMD的方法。

Recent innovations in laser technology have resulted in a remarkable advancement in the generation and application of ultrashort laser pulses. Femtosecond lasers are now used by scientists to "watch" a molecule fall apart as it undergoes a chemical reaction, and by surgeons to perform laser surgery with unprecedented precision. Ultrafast laser technology is still advancing at a rapid rate, and ultimately short pulses may soon be used to perform laser eye surgery with sub-micron precision. Our long-term goal is to increase our capability to image the eye, for improved quality of eye health of Canadians. To achieve this goal, one technological challenge is to be able to clearly image the extremely small "cuts" produced by the femtosecond laser. This requires real-time three-dimensional imaging of structures within the eye with sub-micron spatial resolution. We propose here to simultaneously use multiple (multi-modality) nonlinear optical microscopy methods, to obtain more comprehensive information of the sample. One important method, named epidetected Coherent Anti-Stokes Raman Scattering (e-CARS) microscopy, is an ideal method to realize such ultimate imaging. e-CARS microscopy provides a unique approach to imaging biological samples by using molecular vibrations as a contrast mechanism. e-CARS microscopy is capable of three-dimensional imaging of minute structures with sub-micron resolution, and can be easily implemented into laser surgical systems, since the same optics used in laser surgery can be used for e-CARS microscopy. Thus, in vivo e-CARS microscopy will greatly contribute to the enhanced precision of femtosecond laser eye surgery. Another potentially important application is the diagnosis and follow-up of age-related macular degeneration (AMD). This retinal disease is the leading cause of legal blindness in Canada. Approximately 25% of Canadians over 75 years of age are affected to some level. The development and application of e-CARS microscopy will contribute to the better understanding of the pathophysiology of this disease, and will help to find a cure for AMD.