Department of Physics and Astronomy, Texas A&M University, College Station, TX 77843, United States of America.
National Research Council, AFRL, JBSA Fort Sam Houston, TX 78234, United States of America.
J Photochem Photobiol B. 2021 Sep;222:112271. doi: 10.1016/j.jphotobiol.2021.112271. Epub 2021 Jul 23.
Photobiomodulation (PBM) describes the use of low irradiance light in the red to near-infrared wavelength range to stimulate biological effects in tissue, and many biological and spectroscopic techniques are used to study PBM. However, these techniques focus on the products or downstream effects rather than the electronic transitions that initiate the PBM processes. This study presents a novel approach to studying low irradiance light exposures on individual proteins and/or protein complexes by combining a continuous wave (CW) laser diode with femtosecond transient absorption spectroscopy (TAS), coined here as CW-TAS, and tests the system on reduced cytochrome c (Cyt c) for proof of principle. TAS was conducted using a 532-nm excitation pump beam and a 350-600 nm supercontinuum probe. CW laser diodes with wavelengths of 450 nm, 635 nm, and 808 nm were interchangeably fiber coupled into the HELIOS Fire. Samples of Cyt c were tested by TAS using a pump power of 15 μW, both with and without CW exposure. CW exposures were carried out with irradiances of 1.60 and 3.20 mW/cm, except for 808 nm, which was only tested at 1.60 mW/cm. Both kinetic and global analyses were performed on the TAS data and the time constants for sets with and without CW exposures were compared. The TAS data for Cyt c with the full dosage of CW exposures did not alter the TAS data distinguishably from the control data. No new electronic transient signals were observed beyond the background when testing Cyt c with the CW exposures. Kinetic analysis confirmed that existing transients did not deviate beyond uncertainty. Global time constants for Cyt c were calculated to be 0.25 ± 0.03 ps and 5.1 ± 0.3 ps for the control study, and the time constants for the CW exposed Cyt c were not significantly different. This study concludes that CW irradiation, at doses delivered, does not alter the transient absorption data of Cyt c. The CW-TAS method provides a new tool for studying PBM effects in other proteins and protein complexes that might respond to the CW wavelengths, such as Complex IV, in future studies.
光生物调节（PBM）描述了使用低辐照度的光在红色到近红外波长范围内刺激组织中的生物效应，并且许多生物和光谱技术被用于研究 PBM。然而，这些技术侧重于产物或下游效应，而不是启动 PBM 过程的电子跃迁。本研究提出了一种新的方法，通过将连续波（CW）激光二极管与飞秒瞬态吸收光谱（TAS）结合，研究单个蛋白质和/或蛋白质复合物对低辐照度光的暴露，这里称为 CW-TAS，并在还原细胞色素 c（Cyt c）上进行了系统测试，以验证原理。TAS 使用 532nm 激发泵浦光束和 350-600nm 超连续谱探针进行。450nm、635nm 和 808nm 的 CW 激光二极管可互换地光纤耦合到 HELIOS Fire 中。使用 15μW 的泵浦功率，对 Cyt c 的样品进行 TAS 测试，同时进行和不进行 CW 暴露。除了 808nm 之外，在 1.60 和 3.20mW/cm 的辐照度下进行 CW 暴露，仅在 1.60mW/cm 下测试 808nm。对 TAS 数据进行了动力学和全局分析，并比较了有和没有 CW 暴露的数据的时间常数。进行了完整剂量 CW 暴露的 Cyt c 的 TAS 数据与对照数据没有明显区别。在使用 CW 暴露测试 Cyt c 时，除了背景之外，没有观察到新的电子瞬态信号。动力学分析证实，现有的瞬态没有超出不确定度。计算 Cyt c 的全局时间常数为 0.25±0.03ps 和 5.1±0.3ps 用于对照研究，并且 CW 暴露的 Cyt c 的时间常数没有显著差异。本研究得出结论，在给予的剂量下，CW 照射不会改变 Cyt c 的瞬态吸收数据。CW-TAS 方法为研究可能对 CW 波长（如复合物 IV）有反应的其他蛋白质和蛋白质复合物中的 PBM 效应提供了一种新工具。
