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

这个小企业创新研究计划（SBIR）第一阶段项目将改进多光子显微镜，特别是双光子激发荧光（TPEF）。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，因为它可以减轻散射，穿透深度可以达到1毫米，并且激发波长的影响最小（近红外波长对生命系统的危害明显较小）。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。通过快速测量脉冲特性，在原位，在成像的样品本身，利用这些信息来完美地调整脉冲特性在成像点，在原位，在样品本身，样品设置时间可以提高，图像可以使更亮，更高的对比度和更好的一致性。这既适用于脉冲宽度为70-100秒的商用成像系统，也适用于脉冲宽度为50秒或更短的家用成像系统。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，更快地产生更高质量的TPEF图像，具有更好的一致性。该项目的更广泛的影响/商业潜力是开发仪器和控制系统，通过允许生产高对比度飞秒脉冲，促进超快激光器在新的重要应用中的改进效用。这些脉冲在空间和时间上受到变换限制，这不仅影响成像，而且影响飞秒激光器的所有商业应用。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，被广泛应用于微流控平台和白内障手术等行业，如飞秒激光辅助白内障手术和激光辅助原位角膜磨圆术（LASIK）手术。目前的技术只促进了焦点和相互作用区域之前的脉冲优化。事实上，目前的飞秒激光眼科手术是基于20年前的技术。随着工业开始重新利用改进的飞秒激光器的优势，新的脉冲增强工具，如本文提出的，将是其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.