Botanisches Institut, Universität Düsseldorf, D-4000, Düsseldorf, Fed. Rep. Germany.
Photosynth Res. 1985 Mar;6(1):73-86. doi: 10.1007/BF00029047.
Due to the optical density of photosynthetic tissues the spectral characteristics of fluorescence emitted at 77 K directly from frozen plant material are distorted by differential re-absorption of the emitted light: the emission band related to PSII can be lowered by more than 80%, relative to the PSI band and the profile of the excitation spectra becomes flattened. It is demonstrated that such distortion cannot be neglected as its extent varies from sample to sample. A technique is introduced to eliminate sample artifacts related to self-absorption: subcellular small particles are prepared from rapidly cooled leaves and then 'diluted' without re-thawing at a concentration corresponding to about 5 μg chlorophyll·cm(-3) into a matrix consisting of ice and quartz particles. The photochemical pigment apparatus is expected to remain fixed in the in vivo state. Different kinds of plant material is used and it is demonstrated how this preparative approach allows to study the in vivo distribution of energy between the two photosystems from pure 77 K spectrofluorimetry, even when the optical properties of whole leaves or thalli normally would exclude quantitative analysis.
由于光合组织的光密度，直接从冷冻植物材料在 77K 发出的荧光的光谱特性会受到发射光的差异再吸收的影响而发生扭曲：与 PSI 带相比，与 PSII 相关的发射带可以降低 80%以上，激发光谱的轮廓变得平坦。事实证明，这种扭曲不能被忽视，因为它的程度因样本而异。本文介绍了一种消除与自吸收相关的样品伪影的技术：从小叶片中制备亚细胞小颗粒，然后在不重新解冻的情况下，在浓度约为 5μg 叶绿素·cm(-3)的条件下将其“稀释”到由冰和石英颗粒组成的基质中。预计光化学色素装置将保持在体内状态下固定。使用了不同种类的植物材料，并证明了这种制备方法如何允许从纯 77K 荧光光谱学研究两个光系统之间的能量在体内的分布，即使整个叶片或藻体的光学性质通常会排除定量分析。
