Tailoring confocal microscopy for in-cell photophysiology studies

Abstract

Summary Photoautotrophs environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here we describe a new approach for single-cell and subcellular photophysiology based on the customisation of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to: i. reassess the specialisation of photosynthetic activities in developing tissues of non-vascular plants; ii. identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which are consolidating metabolic connections with their animal hosts, and iii. testify to the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy. Motivation Visualising photosynthetic responses in 3D is essential for understanding most acclimation processes, as light changes within photosynthetic tissues as it penetrates the absorbing/diffusing layers of the cells. To achieve this goal, we developed a new imaging workflow merging confocal microscopy and saturating pulse chlorophyll fluorescence detection. This method applies to samples characterised by increasing complexity and its simplicity will contribute to its widespread use in plant and microalgae photoacclimation studies.

Plant phenotyping relevance

共焦点顕微鏡と飽和パルス式クロロフィル蛍光検出を統合し、単細胞・組織内の光合成生理応答を取得する新規イメージング手法を開発しているため、植物フェノタイピング手法が中心である。

Code and data availability

The supplied blocks describe a custom 3D-Pulse confocal fluorimeter and Fiji/3DSlicer/R analysis, but contain no public deposit, repository, or availability statement for the paper's phenotype data, images, code, or supplementary videos/tables. The only URLs are the bioRxiv DOI and the generic R project site, neither a

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