Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range
Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range
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DOI：
10.21769/bioprotoc.3498
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发表时间：
2020-01-20
期刊：
影响因子：
0.8
通讯作者：
Hughes, Thomas E.
中科院分区：
文献类型：
--
作者：
Drobizhev, Mikhail;Molina, Rosana S.;Hughes, Thomas E.
双光子激光扫描显微镜（2PLSM）是一种最先进的技术，用于组织内部深处的非侵入性成像，具有高3D分辨率，最小的离焦光损伤和最小的自体荧光背景。为了在2PLSM中最佳地应用荧光探针，必须表征以绝对横截面表示的其双光子吸收（2PA）光谱。在最佳波长下激发将使得有可能降低激光功率，从而使光损伤最小化。获得2PA光谱和横截面需要校正双光子激发的荧光信号的激光特性的组合，包括光束空间分布，脉冲持续时间，和绝对功率，在每个波长的调谐范围。为了避免绝对测量方法中所需的繁琐的日常激光表征，通常使用基于独立表征的2PA参考标准的相对方法。通过仔细分析现有的文献数据，我们选择了最可靠的标准的2PA光谱形状和截面测量。在这里，我们描述了一个协议，用于测量的2PA光谱形状和截面的荧光蛋白和其他荧光团与相对荧光法使用这些参考标准。我们的协议首先描述了如何建立一个光学系统，然后如何执行测量。在我们的方案中，我们使用香豆素540 A在二甲基亚砜和LDS 798在氯仿中的光谱形状测量，以覆盖从680到1300 nm的范围，和罗丹明590在甲醇和黄绿素在碱性水（pH 11）的绝对双光子截面测量。
Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for noninvasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in absolute cross sections must be characterized. Excitation at optimum wavelength will make it possible to reduce the laser power and therefore minimize photodamage. Obtaining 2PA spectra and cross sections requires correcting the two-photon excited fluorescence signals for a combination of laser properties, including the beam spatial profile, pulse duration, and absolute power, at each wavelength of the tuning range. To avoid such tedious day-to-day laser characterization required in the absolute measurement method, a relative method based on independently characterized 2PA reference standards is often used. By carefully analyzing the available literature data, we selected the most reliable standards for both the 2PA spectral shape and cross section measurements. Here we describe a protocol for measuring the 2PA spectral shapes and cross sections of fluorescent proteins and other fluorophores with the relative fluorescence method using these reference standards. Our protocol first describes how to build an optical system and then how to perform the measurements. In our protocol, we use Coumarin 540A in dimethyl sulfoxide and LDS 798 in chloroform for the spectral shape measurements to cover the range from 680 to 1300 nm, and Rhodamine 590 in methanol and Fluorescein in alkaline water (pH 11) for the absolute two-photon cross section measurements.