Biophysics Division, University of Illinois, 156 Davenport Hall, 607 S. Mathews, 61801, Urbana, Illinois, USA.
Photosynth Res. 1990 Dec;26(3):181-93. doi: 10.1007/BF00033131.
A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the 'double-flash' kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439-448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 μs) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% Fmax) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 μs after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.
一种新开发的现场便携式多闪光动力学荧光仪，用于测量完整植物叶片中光合作用系统 2 的氧化和还原侧的微秒至毫秒反应的动力学，该仪器已得到描述和验证。该仪器技术是对“双闪光”动力学荧光仪（Joliot 1974 Biochim Biophys Acta 357: 439-448）中使用的技术的改进，其中使用低强度短持续时间的光脉冲来测量饱和单周转光脉冲后荧光产率的变化。本仪器使用快速的短持续时间（2 μs）脉冲序列，以解析每个光激发脉冲后荧光产率变化的完整微秒至毫秒时间尺度动力学轨迹。使用光纤矩阵的差分光学技术允许非常高的灵敏度（噪声水平约为 0.05% Fmax），从而消除了信号平均的需要，并大大降低了进行测量所需的光强度。因此，测量脉冲的光激活作用较小，并且一个完整的多点轨迹（七个点）激发的叶片中的反应中心不到 1%。此外，通过将光激发和测量脉冲光组合在光纤网络中，可以补偿光激发脉冲的尾部，从而可以在光激发后 20 μs 内快速测量事件。这种分辨率使得在野外条件下对完整植物叶片中氧气释放复合物的微秒周转率动力学进行常规测量成为可能。该仪器通过观察闪光数依赖性和抑制剂对闪光诱导荧光瞬变的诱导和衰减动力学的敏感性来进行演示。从这些轨迹中，可以观察到与两电子门的周转率相关的周期二振荡和与氧气释放复合物的周转率相关的周期四振荡。该仪器的应用可扩展到对叶绿体功能的整体植物认识、环境胁迫对植物的影响、除草剂等，以及可能对突变体筛选的应用。
