In vivo tracking of harmonic nanoparticles (HNPs) in living animals is a technique not yet exploited, despite the great potential offered by these markers, due to a lack of an appropriate tool. The main drawback is the necessity to excite nonlinear effects in the millimeter area in a widefield mode with a sufficient signal to noise ratio. Our approach to this problem consists in a redesign of the laser space parameters in a region of high energy per pulse and low repetition rate in the kHz regime, in counter-trend with the actual microscope research technology. We realise this by means of a regeneratively amplified fs-laser system, creating an easy alignable and reproducible Tunable hIGh EneRgy (TIGER) widefield microscope. This one is successfully applied for HNPs tracking in the blood flow of the heart system of a Drosophila larvae, a powerful platform to study socially relevant diseases, such as congenital heart defects in human beings. It is possible to follow nonlinear emitting marker in a remarkable field-of-view of up to 1.5 × 1.5 mm² at 70 frame per seconds. The impact of the energy per pulse, the pulse repetition rate as well as of the photon energy on the SNR is determined and the optimum setup conditions are deduced. At the same time, wavelengths of fundamental and harmonic pulses are carefully considered and tailored to match the transmission fingerprint of the Drosophila larvae. Our findings clearly demonstrate the large impact of precise pulse parameter management in the view of the optical features of the sample, the optical setup and the photosensitivity of the detector. A step-by-step instruction for more general use of the technique is described, opening the path for addressing biological research questions that require far-field imaging at high frame rates with exceedingly high spatial and temporal precision.

中文翻译：

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谐波纳米粒子的体内跟踪：基于 TIGER 宽场显微镜的研究 [邀请]

尽管这些标记提供了巨大的潜力，但由于缺乏适当的工具，在活体动物体内跟踪谐波纳米粒子 (HNP) 是一项尚未开发的技术。主要缺点是必须在具有足够信噪比的宽场模式下在毫米范围内激发非线性效应。我们解决这个问题的方法包括在每脉冲高能量和 kHz 范围内的低重复率区域重新设计激光空间参数，与实际显微镜研究技术相反。我们通过再生放大 fs 激光系统实现了这一点，创建了一个易于对齐和可重复的可调谐高能 (TIGER) 宽视野显微镜。这个成功地应用于 HNPs 跟踪心脏系统的血流果蝇幼虫，一个研究社会相关疾病（例如人类先天性心脏缺陷）的强大平台。可以以每秒 70 帧的速度在高达 1.5 × 1.5 mm ²的非凡视场中跟踪非线性发射标记。确定每个脉冲的能量、脉冲重复率以及光子能量对 SNR 的影响，并推导出最佳设置条件。同时，基波和谐波脉冲的波长经过仔细考虑和定制，以匹配果蝇的传输指纹幼虫。我们的研究结果清楚地表明，从样品的光学特征、光学设置和探测器的光敏性来看，精确脉冲参数管理的巨大影响。描述了该技术更普遍使用的分步说明，为解决生物研究问题开辟了道路，这些问题需要以极高的空间和时间精度以高帧率进行远场成像。