SBIR Phase I: Quantitative Space-time Control for High Contrast Multiphoton Microscopy

这个小型企业创新研究计划(SBIR)第一阶段项目将对多光子显微镜，特别是双光子激发荧光(TPEF)进行改进。这项技术从根本上改变了进行定量生物学研究的方式。它现在是动态、高分辨率(亚微米)研究的首选工具，因为散射减少，穿透深度可以达到1毫米，激发波长的影响最小(近红外波长对生命系统的危害要小得多)。然而，限制TPEF成像应用的挑战是设置时间、图像优化和一致性。通过在成像样品本身内的现场快速测量脉冲特性，使用该信息来在样品本身内的成像点处在现场完美地调整脉冲特性，可以改进样品设置时间，并且可以使图像更明亮、更高对比度和更好的一致性。这适用于使用70-100飞秒脉冲宽度的商业成像系统，以及使用50飞秒或更小脉冲宽度的自制系统。该项目将开发一种新颖的插入式设备，可用于任何多光子显微镜系统，使生物学家能够实现完全自动化的样品设置，以更快、更好的一致性产生更高质量的TPEF图像。该项目更广泛的影响/商业潜力是开发仪器和控制系统，通过允许产生高对比度飞秒脉冲，促进超快激光用于新的重要应用。这些脉冲在空间和时间上受到变换的限制，这不仅影响了成像，而且影响了飞秒激光的所有商业应用。例如，等离子体介导的消融利用放大的飞秒脉冲，是一种强大的过程，广泛应用于各种行业，包括微流控平台和白内障手术，如飞秒激光辅助白内障手术和激光辅助原位角膜磨镶术(LASIK)手术。目前的技术仅便于在聚焦和相互作用区域之前进行脉冲优化。事实上，现在的飞秒激光眼科手术是基于20年前的技术。随着各行各业开始重新利用改进的飞秒激光的好处，新的脉冲增强工具，如这里提出的，将是成功实施的必要条件。

This Small Business Innovation Research Program (SBIR) Phase I project will develop improvements to multiphoton microscopy, especially two-photon excitation fluorescence (TPEF). This technology has radically changed the manner in which quantitative biological studies are performed. It is now the tool of choice in dynamic, high-resolution (sub-micrometer) studies because scattering is mitigated, penetration depths can reach 1 mm, and impact of the excitation wavelength is minimized (near-infrared wavelengths are significantly less harmful to living systems.) However, challenges that limit application of TPEF imaging are setup time, image optimization and consistency. By rapidly measuring pulse characteristics, in situ, within the imaged sample itself, using this information to perfectly adjust pulse characteristics at the imaged point, in situ, within the sample itself, sample setup times can be improved and images can be made to be brighter, higher contrast and have better consistency. This holds for both commercial imaging systems using pulse width ranges from 70-100 fs, as well as home-built systems using pulse widths of 50 fs or less. This project will develop a novel, drop-in device, useable on any multiphoton microscopy system that will enable completely automated sample setup for the biologist to produce higher quality TPEF images faster, with better consistency. The broader impact/commercial potential of this project is the development of instrumentation and control systems to facilitate the improved utility of ultrafast lasers for new and important applications by allowing the production of high-contrast femtosecond pulses. These pulses are transform-limited in space and time, which not only impacts imaging, but all commercial applications of femtosecond lasers. For example, plasma-mediated ablation utilizes amplified femtosecond pulses and is a powerful process that is incorporated in a broad range of industries includes microfluidic platforms, and cataract surgeries, such as femtosecond laser assisted cataract surgery as well as Laser-Assisted in Situ Keratomileusis (LASIK) procedures. Current technologies only facilitate pulse optimization prior to the focus and interaction region. In fact, present femtosecond laser eye-surgery is based on twenty-year old technology. As industries begin to re-tool to take advantage of the benefits of improved femtosecond lasers, new pulse enhancement tools, such as proposed here, will be imperative to their successful implementation.