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首页> 外文会议>Multiphoton microscopy in the biomedical sciences XVII >Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins
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Optimizing ultrafast wide field-of-view illumination for high-throughput multi-photon imaging and screening of mutant fluorescent proteins

机译：针对高通量多光子成像和突变型荧光蛋白的筛选，优化超快速宽视野照明

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摘要

Fluorescence induced by lwo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore's nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.
机译：由低光子吸收（2PA）和三光子吸收（3PA）诱导的荧光正成为深层组织显微镜越来越重要的工具，尤其是与遗传编码的功能探针（例如荧光蛋白（FPs））结合使用时。不幸的是，众所周知，FPs的多光子激发效率很低，并且由于生物荧光团的非线性光学性质与其分子结构之间的关系固有地很复杂，因此没有可行的方法可以提高当前FPs的性能。 。在这里，我们描述了一种新颖的方法，其中我们应用定向进化来优化EGFP的2PA特性。该方法成功的关键在于对突变体的高通量筛选，这将允许选择在广阔的近红外激发波长范围内具有有前途的2PA和3PA特性的变异体。为此，我们构建并测试了宽视场（FOV）飞秒成像系统，然后将其用于量化数以万计的大肠杆菌菌落在550- 1600 nm范围内的多光子激发荧光。在标准的10厘米直径培养皿中表达随机突变的FP。我们提出了目前限制飞秒多光子筛选技术最大通量的不同因素的定量分析，并且还报告了绝对2PA和3PA横截面光谱的定量测量。

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