F1000 reviews about the paper:
Technische Universität München, Germany
This paper provides an excellent and easy method to simultaneously visualize tissue morphology and gene expression patterns with cellular resolution in fixed plant tissue using confocal microscopy. The authors substantially improved a previously known method for whole-mount staining of cell outlines to allow the analysis of all Arabidopsis organs at all stages of development. As this method can be combined with GUS staining, a detailed analysis of morphology and gene expression patterns with a single-cell resolution can be obtained. This method can, for example, provide a basis for the generation of a digital atlas, combining organ architecture and gene expression patterns in plants.
Xing Wang Deng
Yale University, United States of America
High definition goes green. In this article, Truernit et al. present a plant-tissue staining method that, with the help of a confocal microscope, produces high-resolution images and 3D reconstruction of the cellular organization of plant organs without production of tissue sections. The method also enables 3D imaging of gene expression with single-cell precision. I strongly suggest taking a closer look at the beautiful images and supplemental movies made available by the authors.
Here is the paper abstract:
Plant Cell. 2008 Jun 3.
High-Resolution Whole-Mount Imaging of Three-Dimensional Tissue Organization and Gene Expression Enables the Study of Phloem Development and Structure in Arabidopsis.
Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauqui JC.
Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, Unité de Recherche 501, Institut National de la Recherche Agronomique, 78026 Versailles cedex, France.
Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.
The key is hot methanol. The following is the detailed methods section: mPS-PI Staining Imaging of Sieve Plates and Aniline Blue Staining Confocal Microscopy Data Processing
Tissue was immersed in GUS staining solution (100 mM sodium phosphate buffer, pH 7.2, 10mMsodium EDTA, 0.1% Triton X-100, and 1 mg/mL 5-bromo-4-chloro-3-indolyl-D-glucuronic acid [Duchefa], to which potassium ferrocyanide and potassium ferricyanide to a final concentration of 2.5 mM were freshly added). The staining solution was infiltrated into the tissue by subjecting samples to a vacuum for 2 32 min. Samples were incubated at 378C for defined times, depending on the marker used (Bauby et al., 2007), and then rinsed with water. PS-PI staining followed.
Whole seedlings or plant organs were fixed in fixative (50%methanol and 10% acetic acid) at 48C for at least 12 h. Tissue could also be stored in the fixative for up to 1 month. The tissue was then transferred to 80% ethanol and incubated at 808C for 1 to 5 min, depending on tissue type (for example leaves, 1min; floral stalks, 5min). Tissue was transferred back to fixative and incubated for another hour. Next, tissue was rinsed with water and incubated in 1% periodic acid at room temperature for 40 min. The tissue was rinsed again with water and incubated in Schiff reagent with propidium iodide (100 mM sodium metabisulphite and 0.15 N HCl; propidium iodide to a final concentration of 100 mg/mL was freshly added) for 1 to 2 h or until plants were visibly stained. The samples were then transferred onto microscope slides and covered with a chloral hydrate solution (4 g chloral hydrate, 1 mL glycerol, and 2 mL water). Slides were kept overnight at room temperature in a closed environment to prevent drying out. Next, excess chloral hydrate was removed and several drops of Hoyer’s solution (30 g gum arabic, 200 g chloral hydrate, 20 g glycerol, and 50 mL water) were placed over the tissue, and a cover slip was placed on top. Slides were left undisturbed for a minimum of 3 d to allow the mounting solution to set. For the staining of roots and emerged lateral roots, the ethanol step was omitted. For staining of ovules and seeds, siliques were harvested and slit open on one side. Tissue was fixed as described above and then subjected to an overnight treatment of 1% SDS and 0.2 N NaOH at room temperature. Siliques were rinsed in water, incubated in 25% bleach solution (2.5% active Cl) for 1 to 5 min, rinsed again, and then transferred to1% periodic acid. The samples were then further processed as described above.
Stem and hypocotyl cross sections (60 to 100 mm thick) were obtained with a vibratome (Leica VT1000S). Samples were then subjected to PS-PI staining as described above. Callose staining was performed as described by Stadler et al. (1995).
A Leica TCS-SP2-AOBS spectral confocal laser scanning microscope (Leica Microsystems) was used. The excitation wavelength for PS-PI–stained samples was 488 nm, and emission was collected at 520 to 720 nm. GUS staining was imaged with the AOBS reflection mode of the confocal microscope. The excitation wavelength was 488 nm, and the reflection signal was collected between 485 and 491 nm. Callose fluorescence was collected between 480 to 515 nm using a 405-nm laser.
Data were processed for some two-dimensional orthogonal sections, 3D rendering, and movie exports using the open source software Osirix (Rosset et al., 2004; http://homepage.mac.com/rossetantoine/osirix/) on a quadxeon 2.66-Ghz, 2-GB RAM Apple Mac pro workstation. RGB stacks of confocal images were imported as DICOm files into Osirix prior to surface rendering. For the production of optical sections, for signal quantification, and for cell length measurements, we used the Leica Confocal Software version 2.61.
Imaging of Sieve Plates and Aniline Blue Staining
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