Optical Coherence Microscopy. A Technology for Rapid, in Vivo, Non-Destructive Visualization of Plants and Plant Cells,

Abstract

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 m in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 ϫ 5 ϫ 10 m) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a threedimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.Studies in plant physiology and development characteristically follow changes in space and time that occur as part of normal plant activity or in response to exogenous stimuli. Typical studies require the destruction and analysis of a plant or a tissue sample, followed by the collection and analysis of a second distinct plant or sample. Thus, biological responses or changes are inferred by comparing different plants or samples. Such approaches have been used for centuries and have produced a great deal of knowledge. However, when scientists are able to nondestructively follow biological changes, important concepts and insights have emerged. For example, critical genes involved in programmed cell death were found in Caenorhabditis elegans partially because the developing nematode is nearly transparent, allowing the fate of each cell to be followed in vivo by light microscopy (Gilbert, 1998). Similarly, an elegant fate map for Arabidopsis roots was constructed because the relatively transparent roots allow changes in individual plants to be followed continuously (Dolan et al., 1993). This study led to new discoveries such as the presence of downward communication between mature root cells and the root apical meristem and short-range control of differentiation signals (van den Berg et al., 1997a(van den Berg et al., , 1997b.Except for the relatively transparent Arabidopsis root, plants provide a challenge for in vivo an...

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