Leaf Structure and Function in Four Dimensions: Non-invasive MicroCT Imaging During Gas-exchange Measurements
Authors: Tholen Danny , Scheffknecht Susanne , Voggeneder Klara , Weiss Elisabeth , Théroux-Rancourt Guillaume 
Published: March 2022

&lt;p&gt;&lt;span&gt;Plant physiologists have used microscopy to study how leaf anatomy is related to photosynthetic performance and how this relation is affected by environmental conditions. However, leaf anatomy is not invariant over time: small pores on the leaf surface (stomata) open and close within minutes in response to the availability of water, CO&lt;/span&gt;&lt;sub&gt;&lt;span&gt;2&lt;/span&gt;&lt;/sub&gt;&lt;span&gt; and light. Within tens of minutes following a water deficit, cells in &lt;/span&gt;&lt;span&gt;many&lt;/span&gt;&lt;span&gt; leaves also shrink significantly in volume and the leaf undergoes structural changes as a result of wilting. Gas-exchange setups can monitor changes in photosynthesis and transpiration under such conditions, but classical microscopy techniques are not well-suited to capture the concomitant changes in leaf anatomy &lt;/span&gt;&lt;span&gt;for two main reasons&lt;/span&gt;&lt;span&gt;. &lt;/span&gt;&lt;span&gt;First, available non-destructive microscopy techniques are limited in resolution and imaging depth, making it difficult to analyze changes in anatomy to the required detail. Second, u&lt;/span&gt;&lt;span&gt;sing sectioned fixated samples is known to be associated with tissue shrinkage, swelling or deformation, making estimates of cellular volumes and surfaces prone to art&lt;/span&gt;&lt;span&gt;i&lt;/span&gt;&lt;span&gt;facts. &lt;/span&gt;&lt;span&gt;Moreover, t&lt;/span&gt;&lt;span&gt;he destructive nature of &lt;/span&gt;&lt;span&gt;these&lt;/span&gt;&lt;span&gt; techniques makes it impossible to monitor changes in leaf anatomy during ongoing gas-exchange measurements. These limitations hinder advancing our understanding of the relation between leaf anatomy and photosynthesis &lt;/span&gt;&lt;span&gt;or&lt;/span&gt;&lt;span&gt; transpiration.&lt;/span&gt;&lt;/p&gt;&lt;p&gt;&lt;span&gt;Here, we present a novel gas-exchange setup that combines synchrotron-base&lt;/span&gt;&lt;span&gt;d &lt;/span&gt;&lt;span&gt;high-resolution computed tomography (microCT) with &lt;/span&gt;&lt;span&gt;c&lt;/span&gt;&lt;span&gt;oncurrent measurements of gas-exchange using an commercially available infra-red gas analyzer. We designed &lt;/span&gt;&lt;span&gt;and constructed &lt;/span&gt;&lt;span&gt;a novel gas-exchange cuvette with CO&lt;/span&gt;&lt;sub&gt;&lt;span&gt;2&lt;/span&gt;&lt;/sub&gt;&lt;span&gt; and H&lt;/span&gt;&lt;sub&gt;&lt;span&gt;2&lt;/span&gt;&lt;/sub&gt;&lt;span&gt;O control that allow&lt;/span&gt;&lt;span&gt;s&lt;/span&gt; &lt;span&gt;for&lt;/span&gt;&lt;span&gt; non-invasive monitoring of leaf anatomy in a microCT setup. Custom-built sensors were used to &lt;/span&gt;&lt;span&gt;measure&lt;/span&gt;&lt;span&gt; light intensity and leaf temperature. At given time points &lt;/span&gt;&lt;span&gt;during gas-exchange measurements&lt;/span&gt;&lt;span&gt;, 300-500 X-ray projections &lt;/span&gt;&lt;span&gt;(&lt;/span&gt;&lt;span&gt;100 ms&lt;/span&gt;&lt;span&gt;)&lt;/span&gt;&lt;span&gt; were taken while the chamber rotated 180&amp;#176;. &lt;/span&gt;&lt;span&gt;From this data, a&lt;/span&gt;&lt;span&gt; leaf volume corresponding to 0.5 mm&lt;/span&gt;&lt;sup&gt;&lt;span&gt;2&lt;/span&gt;&lt;/sup&gt;&lt;span&gt; leaf surface was reconstructed &lt;/span&gt;&lt;span&gt;at high resolution (&lt;/span&gt;&lt;span&gt;0.325 &amp;#181;m &lt;/span&gt;&lt;span&gt;per &lt;/span&gt;&lt;span&gt;voxel edge). &lt;/span&gt;&lt;/p&gt;&lt;p&gt;&lt;span&gt;The setup provides 3D images that can be used to measure the aperture of multiple stomata and the volumes, shapes and surface areas of cells and airspaces within the leaf. We found that the same leaf section can be scanned several times without measurable radiation damage, allowing for the combination of &lt;/span&gt;&lt;span&gt;three&lt;/span&gt;&lt;span&gt; spatial dimensions with time to create a 4D analysis of the leaf structure. Using poplar, willow and &lt;/span&gt;&lt;span&gt;&lt;em&gt;Arabidopsis&lt;/em&gt;&lt;/span&gt;&lt;span&gt; leaves we studied how leaf anatomy rapidly adjusts after limiting water availability and show that such effects are not limited to the stomatal pore alone. We discuss the issues and pitfalls with the methodology and suggest avenues for future improvement.&lt;/span&gt;&lt;/p&gt;