Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It.
Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It.
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DOI：
10.3390/ijms23020770
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发表时间：
2022-01-11
影响因子：
5.6
通讯作者：
Franklin J
中科院分区：
文献类型：
--
作者：
Drobizhev M;Molina RS;Franklin J
红色荧光蛋白和基于它们的生物传感器对于双光子激光显微镜（TPLM）是潜在有益的，因为与绿色荧光蛋白相比，它们可以对更深层的组织成像。然而，一些出版物报道了它们非常快的光漂白，特别是在750-800 nm激发时。在这里，我们研究了mCherry，mPlum，tdTomato和jREX-GECO 1的多光子漂白特性，测量了整个双光子吸收光谱中不同激发波长下光漂白速率K的功率依赖性。虽然这些蛋白质都含有相同化学结构的发色团，但它们的多光子漂白机制却各不相同。启动光化学反应所需的光子数根据波长和功率而变化，从2（所有四种蛋白质）到3（jREX-GECO 1）到4（mCherry，mPlum，tdTomato），甚至高达8（tdTomato）。我们发现，在足够低的激发功率P下，速率K通常遵循二次幂相关，当功率超过特定阈值P* 时，这种二次幂相关转变为高阶（K~Pα，α > 2）。TPLM的最佳强度接近P*，因为它提供最高的信号背景比，并且激光强度的任何进一步降低都不会改善荧光/漂白速率比。此外，应避免使用短于特定阈值的波长，以避免由于多光子电离而导致的快速漂白。
Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.
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