Multi-wavelength femtosecond laser sources for intravital multiphoton microscopy

描述（由申请人提供）：这是一个合作研究项目，汇集了光学技术组（徐博士）和体内成像组（林博士）。这两个小组有一个共同的目标，即开发成像技术来解决生物医学问题和满足临床需求。在这里，我们集中在需要改善造血干细胞（HSC）归巢和移植后造血干细胞（HSCT）。这种挽救生命的手术通常是治愈血液系统癌症（如白血病或淋巴瘤）患者的最后希望，但只有当足够数量的移植HSC能够到达并植入患者的骨髓（BM）时，才能实现成功的移植。为了帮助改善干细胞归巢和植入，Lin团队开发了活体成像方法来跟踪移植后活动物BM中的单个HSC。然而，由于现有成像技术的不足，BM微环境的当前视图受到严重限制。为了更全面地了解BM微环境，其中多种细胞类型相互作用并形成HSC植入的支持性生态位，Xu小组将开发一种用于非线性显微镜的新型基于纤维的光源，这将使多个荧光指示剂同时成像，并使无标记谐波产生和振动成像成为可能。新光源与活体显微镜的集成将使Lin小组能够继续进行设想的实验，但由于缺乏合适的技术而被搁置。拟议的来源基于以下创新：（1）大模面积（LMA）光纤中的孤子自频移（SSFS）使得能够产生从光纤激光器以电信波长播种的能量的、宽波长可调谐的孤子脉冲，并且随后的基波波长的二次谐波产生（SHG）使得能够实现单个交钥匙、低成本，基于光纤的光源，以产生三个独立的波长可调光源来激发多个荧光团。2)全光纤、高速强度调制，以电子方式控制波长、重复率和脉冲延迟。3)一个单一的光源将使实验，目前需要两个同步钛：蓝宝石激光器加上光学参量振荡器（OPO）和再生放大器。利用为电信行业开发的高度成熟和集成的技术，我们的目标是创建一个真正强大和多功能的“电信级”飞秒源。多功能性对于定制源以满足特定成像需求是重要的，而鲁棒性对于需要对大量动物进行纵向成像的生物学研究是必不可少的。该计划的成功完成不仅将推动成像技术的发展，还将推动干细胞研究的发展。此外，该技术将广泛适用，并将显着增加飞秒源的可及性，以其他生物医学研究人员。

DESCRIPTION (provided by applicant): This is a collaborative research program that brings together an optical technology group (Dr. Xu) and an in vivo imaging group (Dr. Lin). The two groups share a common goal to develop imaging technology for solving biomedical problems and addressing clinical needs. Here we focus on the need to improve hematopoietic stem cell (HSC) homing and engraftment after HSC transplantation (HSCT). This life-saving procedure is often the last hope of cure for patients with cancers of the blood system such as leukemia or lymphoma, but successful transplantation can be achieved only if a sufficient number of transplanted HSCs are able to reach and engraft the patient's bone marrow (BM). To help improve stem cell homing and engraftment, the Lin group has developed intravital imaging methods to track individual HSCs in the BM of live animals after transplantation. However, the current view of the BM microenvironment is severely limited due to the inadequacies of the available imaging technology. To gain a more comprehensive view of the BM microenvironment, where multiple cell types interact and form a supportive niche for HSC engraftment, the Xu group will develop a novel fiber-based source for nonlinear microscopy, which will enable simultaneous imaging of multiple fluorescent indicators as well as enabling label-free harmonic generation and vibrational imaging. Integration of the new source with the intravital microscope will enable the Lin group to proceed with experiments that had been envisioned but were held back due to lack of a suitable technology. The proposed source is based on the following innovations: (1) Soliton self-frequency shift (SSFS) in a large mode area (LMA) fiber enables the generation of energetic, widely wavelength tunable soliton pulses seeded from a fiber laser at the telecom wavelength, and the subsequent second harmonic generation (SHG) of the fundamental wavelength enables a single turn-key, low-cost, fiber-based source to generate three independent wavelength tunable sources to excite multiple fluorophores. 2) All-fiber, high-speed intensity modulation to electronically control the wavelength, repetition rate, and pulse delay. 3) A single light source will enable experiments that currently require two synchronized Ti:sapphire lasers plus an optical parametric oscillator (OPO) and a regenerative amplifier. Leveraging the highly mature and integrated techniques that have been developed for the telecommunications industry, we aim to create a "telecom grade" femtosecond source that is truly robust and versatile. The versatility is important for tailoring the source to meet specific imaging needs while the robustness is essential for the biological studies that require longitudinal imaging of large cohorts of animals. The successful completion of this program will not only advance imaging technology but also advance stem cell research. In addition, the technology will be broadly applicable and will significantly increase the accessibility of femtosecond sources to other biomedical researchers.