In the natural photosynthetic system, the pigment molecules in the light-harvesting antenna absorb the sunlight light energy and transfer the excited state energy to the reaction center to drive the subsequent charge separation and other processes, which usually occur on the time scale of tens of femtoseconds to tens of picoseconds. Thanks to the development of time-resolved spectroscopy, the ultrafast excited state dynamics of photosynthetic systems have been extensively studied. Recent results show that the coupling between the vibrational mode and the electronic state of pigment molecules plays an important role in promoting the efficient energy transfer and charge separation of photosynthetic systems. The special electron vibrational coherent coupling effect is considered to be the key point to reveal the efficient light energy conversion process such as quantum coherent energy transfer in photosynthetic systems, and has become the focus of cutting-edge research in the world. However, there is still a lack of clear and intuitive understanding of the influence of electron-vibration coupling effect on the dynamic evolution of the electronic state of photosynthetic system, and more advanced ultrafast spectral characterization techniques are needed to reveal the physical mechanism behind it.
Fig. 1.(a) Evolution of transient fluorescence spectra of 5×10 mol/L chlorophyll a ethanol solution at 400 nm excitation over time; (b) Transient fluorescence spectra of chlorophyll a at different time delays at excitation wavelengths of 400 nm and (c) 630 nm; (d) (e) The (d) peak position and (e) peak width of transient fluorescence as a function of time when the Soret (400 nm) and Q (630 nm) absorption bands of chlorophyll a are excited, where the dots represent the data points and the curves represent the single exponential fitting results.
Ultrafast time-resolved transient fluorescence spectroscopy is an effective means to study the dynamics of the excited state of photosynthesis, because the detected signals are all from the excited state of electrons, and the analysis of physical processes is simple and straightforward. At present, the fringe camera is the mainstream commercial instrument that can realize broadband transient fluorescence spectroscopy measurement, which was previously monopolized by Hamamatsu of Japan, but due to the limitations of its electronic measurement principle, the time resolution can often only reach picoseconds. However, the energy transfer between photosynthetic pigments can reach the time scale of 100 femtoseconds, and the photodamage threshold is low, so the technical indicators such as time resolution and detection sensitivity are put forward. The research team of Weng Yuxiang and Chen Hailong (SM6 group) of the Institute of Physics, Chinese Academy of Sciences/Beijing National Research Center for Condensed Matter Physics has long been committed to the independent research and development of femtosecond time-resolved non-collinear optical parametric amplification transient fluorescence spectral detection technology, and has achieved high temporal resolution (<80fs), high optical gain (>10), wide spectral measurement bandwidth (>150nm), high detection sensitivity ( <15 photons/pulse) and other excellent technical indicators, reaching the international leading level. Recently, the innovative cone fluorescence collection and ring amplification scheme has greatly suppressed the influence of quantum noise fluctuations in this device, and improved its signal-to-noise ratio by an order of magnitude. This technique was published online in the journal Review of Scientific Instruments (2024, 95, 033008) titled "Femtosecond fluorescence conical optical parametric amplification spectroscopy". And has previously obtained the national invention patent authorization (ZL202110762614.3). The team applied this technique to conduct a series of studies on the dynamics of ultrafast excited states in typical photosynthetic systems, revealing the vibrational energy transfer of chlorophyll a molecules in higher plants in the solution environment and in the excited state of electrons in the light-harvesting antenna LHCII, the energy transfer mechanism of photosynthetic bacterial reaction centers, and the charge separation process.
Fig. 2.(a) Transient fluorescence spectra of chlorophyll a in 400 nm excitation LHCII with different time delays, (b) Transient fluorescence peaks of chlorophyll a in ethanol solution and LHCII with time under 400 nm excitation, (c) Anisotropic attenuation kinetics of chlorophyll a fluorescence in ethanol solution and LHCII, where the dots represent the data points and the curves represent the single exponential fit.
In higher plants, chlorophyll a molecule is the basic pigment unit that constitutes a photosynthetic light-harvesting antenna. Using femtosecond time-resolved broadband fluorescence spectroscopy, the transient fluorescence emission spectra of chlorophyll a molecules in solution were captured for the first time within a few picoseconds after photoexcitation (Fig. 1). Subsequently, it was proved that this anomaly stems from the vibrational cooling process on the excited state of chlorophyll a, that is, the excess vibrational energy caused by the relaxation of the electronic state needs to be dissipated by interaction with the surrounding solvent molecules, which greatly prolongs the lifetime of the higher-order vibrational mode. As a control, the spectral blueshift and narrowing caused by the vibrational cooling of chlorophyll a molecules in a typical higher plant light-harvesting antenna LHCII lasted only 100 femtoseconds (Figure 2), corresponding to the time scale of interpigment energy transfer within the antenna. These results indicate that the excited state high-order vibrational mode of the pigment molecule participates in and promotes the energy transfer inside the photoharvesting antenna, and proposes a new physical image for the key role of the electron-vibrational coupling effect in the photosynthetic energy transfer process.
Fig. 3.(a) Evolution of transient fluorescence spectra of Rps. BRC at 750 nm excitation over time; (b) Transient fluorescence spectra at different time delays, with fluorescence peaks near 820 nm and 900 nm coming from bacterial chlorophyll B and P, respectively; (c) Fluorescence emission kinetics of bacterial chlorophyll B (815 nm) and P (915 nm), where the grey filled curve represents the instrument response function at 750 nm, the dots are the data points, and the curves are the multi-exponential fitting results.
In addition, in the Purple Bacteria Reaction Center (Rps. BRC), the pigment pair P composed of two bacterial chlorophyll a molecules acts as an electron donor to drive the charge separation process of the photosynthetic reaction. Due to the different environments of the pigments, the Qy absorption peaks of chlorophyll H and chlorophyll B of the two monomeric demagnesium demagnesium bacteria are separated from each other and serve as energy donors and electron acceptors for pigment pair P, so they are ideal systems for studying the energy transfer and charge separation process between pigments. Thanks to the femtosecond time resolution and broadband spectroscopy measurement capabilities of the developed transient fluorescence spectroscopy technology, two different transient fluorescence components related to bacterial chlorophyll B and P were directly confirmed. The obtained fluorescence kinetics clearly reveal the ultrafast energy transfer of demagnesium-demagnesium bacterial chlorophyll H to bacterial chlorophyll B (98 fs), bacterial chlorophyll B to pigment pair P (170 fs), and the time scale of charge separation (3.5 ps) (Figure 3). Notably, the fluorescence emission lifetime of bacterial chlorophyll B at sub-200 femtoseconds is expected to be significantly extended to about 400 femtoseconds, suggesting that there may be some coupling between the excited electron state of B and the electron vibration dynamics of P, and has a potential promoting effect on the energy transfer process (Fig. 4). This discovery will help to further understand the mechanism of the influence of electron vibrational coupling on the photoinduced primordial process in the photosynthetic reaction center.
Fig. 4.(a) Schematic diagram of the structure of Rps. BRC, where P represents a pair of strongly coupled bacterial chlorophyll a, B and B represent bacterial chlorophyll a monomers, H and H represent demagnesium-demagnesium bacterial chlorophyll a monomers, and Q and Q represent quinones; （b）Rps. Schematic diagram of the electron state energy transfer (EET) and charge separation (CS) process of BRC after photoexcitation, the fluorescence of bacterial chlorophyll B and P radiation are represented by brown and purple arrows, respectively, where the vibration dynamics of P and the coupling of B electronic states may play a key role in the rapid energy transfer process from B to P.
上述工作基于该团队自主研发的飞秒时间分辨宽带荧光光谱技术，直观地给出了光合体系内色素分子的超快激发态动力学，其结果还可为设计高效的人工模拟光合体系提供支持。 研究结果一以“Unraveling the Excited-state Vibrational Cooling Dynamics of Chlorophyll-a Using Femtosecond Broadband Fluorescence Spectroscopy”为题发表于The Journal of Chemical Physics期刊上；研究结果二以“Primary Processes in Bacterial Reaction Center Revealed by Femtosecond Broadband Fluorescence Spectroscopy”为题发表于Chinese Journal of Chemical Physics期刊上。 中国科学院物理所博士研究生刘鹤元为文章第一作者，物理所陈海龙研究员为通讯作者。 上述研究得到了国家重点研发计划，国家自然科学基金，中国科学院青年科学家基础研究项目，中国科学院战略性先导研究计划，山东省自然科学基金以及综合极端实验条件装置（SECUF）的支持。
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