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，我们重点关注造血干细胞移植（HSCT）后改善造血干细胞（HSC）归巢和植入的需要。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造血干细胞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的归巢和植入，Lin小组开发了活体成像方法来追踪移植后活体动物骨髓中的单个hsc。然而，由于现有成像技术的不足，目前对BM微环境的看法受到严重限制。为了更全面地了解骨髓微环境，其中多种细胞类型相互作用并形成支持HSC植入的生态位，Xu团队将开发一种新型的基于纤维的非线性显微镜源，它将能够同时成像多种荧光指标，并实现无标签谐波产生和振动成像。新光源与活体显微镜的整合将使Lin小组能够继续进行预期的实验，但由于缺乏合适的技术而被推迟。提出的光源基于以下创新：(1)大模区（LMA）光纤中的孤子自频移（SSFS）能够在电信波长的光纤激光器中产生高能、宽波长可调的孤子脉冲，随后的基波二次谐波产生（SHG）使单个交钥匙、低成本、基于光纤的源能够产生三个独立的波长可调源，以激发多个荧光团。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)单个光源将使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）和再生放大器的实验成为可能。利用已经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健壮和通用的“电信级”飞秒源。多功能性对于定制源以满足特定成像需求非常重要，而鲁棒性对于需要对大型动物群体进行纵向成像的生物学研究至关重要。该项目的成功完成不仅将推动成像技术的发展，也将推动干细胞研究的发展。此外，该技术将广泛适用，并将大大增加飞秒源对其他生物医学研究人员的可及性。

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.