Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency.
Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency.
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DOI：
10.1117/1.nph.10.4.044303
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发表时间：
2023-10
期刊：
影响因子：
5.3
通讯作者：
Shtrahman, Matthew
中科院分区：
文献类型：
--
作者：
Saidi, Samir;Shtrahman, Matthew
双光子（2p）显微镜在历史上依赖于钛蓝宝石脉冲激光器，其昂贵且占地面积大。最近，几个制造商已经开发出了较便宜的紧凑型脉冲激光器，其优化用于绿色荧光团的2p激发。然而，缺乏对其质量的定量评价。我们描述了一个简单的方法来系统地评估2p激发效率，经验测量的质量的脉冲激光和它的能力，引起2p诱导荧光。通过测量脉冲宽度，重复率和荧光输出，我们计算了2p激发效率的测量值，并将其与920至930 nm波长范围内的四种商用紧凑型脉冲激光器进行了比较。2p激发效率在所测试的激光器之间变化很大。Coherent Axon显示出最佳的2p激发效率（），超过钛蓝宝石参考激光器（定义为效率= 1）。然而，由于其长脉冲宽度，其测量的荧光是适度的。在紧凑型激光器中，Toptica Femtofiber Ultra表现出测量荧光（）和2p激发效率（）的最佳组合。我们描述了一种简单的方法，激光器开发人员和最终用户都可以使用基准的2p激发效率的激光器用于2p显微镜。
Two-photon (2p) microscopy has historically relied on titanium sapphire pulsed lasers that are expensive and have a large footprint. Recently, several manufacturers have developed less expensive compact pulsed lasers optimized for 2p excitation of green fluorophores. However, quantitative evaluation of their quality is lacking. We describe a simple approach to systematically evaluate 2p excitation efficiency, an empiric measure of the quality of a pulsed laser and its ability to elicit 2p induced fluorescence. By measuring pulse width, repetition rate, and fluorescence output, we calculated a measure of 2p excitation efficiency , which we compared for four commercially available compact pulsed lasers in the 920 to 930 nm wavelength range. 2p excitation efficiency varied substantially among tested lasers. The Coherent Axon exhibited the best 2p excitation efficiency (), exceeding that of a titanium sapphire reference laser (defined to have efficiency = 1). However, its measured fluorescence was modest due to its long pulse width. Of the compact lasers, the Toptica Femtofiber Ultra exhibited the best combination of measured fluorescence () and 2p excitation efficiency (). We describe a simple method that both laser developers and end users can use to benchmark the 2p excitation efficiency of lasers used for 2p microscopy.