Multi-wavelength femtosecond laser sources for intravital multiphoton microscopy

描述(申请人提供)：这是一个合作研究项目，汇集了光学技术小组(徐博士)和活体成像小组(林博士)。这两个小组有一个共同的目标，那就是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，我们着重于改善造血干细胞移植(HSCT)后的归巢和植入的必要性。这种挽救生命的程序往往是白血病或淋巴瘤等血液系统癌症患者治愈的最后希望，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跟踪移植后活体动物骨髓中的单个HSCs。然而，由于现有成像技术的不足，目前对BM微环境的看法严重受限。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支持HSC植入的利基环境，Xu团队将开发一种用于非线性显微镜的新型光纤光源，该光源将实现多个荧光指示剂的同时成像，并实现无标记谐波产生和振动成像。将新的光源与活体显微镜相结合，将使林小组能够继续进行原本设想的实验，但由于缺乏合适的技术而被搁置。提出的光源基于以下创新：(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种子在电信波长处产生的能量高、宽波长可调的孤子脉冲，并且随后的基波的二次谐波产生(SHG)使得单个交钥匙、低成本的基于光纤的光源能够产生三个独立的波长可调谐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、重复频率和脉冲延迟。3)单一光源将实现目前需要两个同步的钛宝石激光器加上一个光学参量振荡器(OPO)和一个再生放大器的实验。利用为电信行业开发的高度成熟和集成的技术，我们的目标是创造一种真正强大和多功能的“电信级”飞秒光源。多功能性对于定制信号源以满足特定的成像需求是重要的，而稳健性对于需要对大量动物队列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促进成像技术的进步，也将促进干细胞研究的进步。此外，这项技术将得到广泛应用，并将显著增加其他生物医学研究人员对飞秒源的可及性。

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.