Abstract
BackgroundThe visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique. Recently, a laboratory-based X-ray microscope adopting the phase contrast CT was developed as a powerful tool for the observation of weakly absorbing biological samples. Here we report the observation of unstained pansy seeds using the laboratory-based X-ray phase-contrast CT.ResultsA live pansy seed within 2 mm in size was simply mounted inside a plastic tube and irradiated by in-house X-rays to collect projection images using a laboratory-based X-ray microscope. The phase-retrieval technique was applied to enhance contrasts in the projection images. In addition to a dry seed, wet seeds on germination with the poorer contrasts were tried. The phase-retrieved tomograms from both the dry and the wet seeds revealed a cellular level of spatial resolutions that were enough to resolve cells in the seeds, and provided enough contrasts to delineate the boundary of embryos manually. The manual segmentation allowed a 3D rendering of embryos at three different stages in the germination, which visualized an overall morphological change of the embryo upon germination as well as a spatial arrangement of cells inside the embryo.ConclusionsOur results confirmed an availability of the laboratory-based X-ray phase-contrast CT for a 3D-structural study on the development of small seeds. The present method may provide a unique way to observe live plant tissues at micron resolutions without structural perturbations due to the sample preparation.
Highlights
The visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique
The electron microscopy that provides a nanometer scale of spatial resolutions can be classified into the transmission electron microscope (TEM) and the scanning electron microscope (SEM) [8]
A promising application of the electron microscopy to plant specimens would be the correlative light and electron microscopy in which the light microscopy and the TEM are used in a complementary manner [9]
Summary
The visualization of internal 3D-structure of tissues at micron resolutions without staining by contrast reagents is desirable in plant researches, and it can be achieved by an X-ray computed tomography (CT) with a phase-retrieval technique. An optical phase-contrast tomography termed “marker-free phase nanoscopy” was developed [7], which enabled an observation of unstained specimens at 90 nm resolution but with a remaining limitation in the sample thickness. A promising application of the electron microscopy to plant specimens would be the correlative light and electron microscopy in which the light microscopy and the TEM are used in a complementary manner [9]. Another perspective is the serial block face SEM in which consecutive SEM images are integrated to reconstruct a large volume with dimensions of hundreds of microns [10, 11]. The electron microscopy is quite useful for the high-resolution observation, it is time consuming and requires a laborious preparation of ultrathin specimens with thicknesses of around 100 nm, due to a low penetration power of electron beams
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