Abstract
Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow for 3D X-ray μCT and associated xylem staining and visualisation techniques, in particular secondary xylem programmed-cell-death (PCD) delay during gelatinous fibre (g-fibre) development. Many of the staining techniques here are transferable to other woody species such as poplar and Eucalyptus.
Keywords: Micro-computed tomography, Tension wood, Cell wall staining, Xylem visualisation, Plant histology
Materials and Reagents
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Glass slides
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Cover slips
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48 well plates
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Razor blades
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Short rotation coppice (SRC) willow trees [cultivar Resolution-pedigree: {S. viminalis. x (S. viminalis. x S. schwerinii SW930812)] x [S. viminalis. x (S. viminalis. x S. schwerinii ‘Quest’)]}
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37% Formaldehyde (36.5-38% in H2O) (Sigma-Aldrich, catalog number: F8775 )
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Acetic acid (≥ 99.7%) (Sigma-Aldrich, catalog number: 320099 )
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Ethanol
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FAA: 3.7% formaldehyde, 5% acetic acid and 47.5% ethanol
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Oasis® floral foam (OASIS Floral Products) (http://oasisfloralproducts.com/)
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Safranin-O solution (Sigma-Aldrich, catalog number: HT90432 )
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DPX Mountant for histology (Sigma-Aldrich, catalog number: 06522 )
Note: This product is named “Slide mounting medium (p-Xylene-bis(N-pyridinium bromide))” at Sigma-Aldrich.
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Distilled water
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β-D-glucosyl Yariv reagent (Biosupplies Australia Pty Ltd., catalog number: 100-2 )
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Histoclear clearing agent (National Diagnostics, catalog number: HS-200 )
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Chlorazol black E/direct black 38 (Sigma-Aldrich, catalog number: C1144 )
Equipment
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Reichert Sliding Microtome
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Tweezers (for handling section)
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Light microscope
Procedure
X-ray μCT can also potentially be used for a broader range of crops, and a more diverse set of biological questions. In most cases, care should be taken to air-dry material prior to scanning; water saturation will reduce cell wall contrast while forced drying beyond fibre saturation point runs the risk of inducing cell collapse, altering the plant’s tissue architecture. An example of this wider potential for the technology in plant research is provided in Figure 1 and Video 1.

Figure 1. Miscanthus internode 3D render. X-ray 3D Computational tomography example images of mature Miscantus x giganteus stem.
Video 1. 3D render of X-Ray μCT scans of willow (cultivar Resolution). 2 cm stem section (debarked). Vessel lumens are coloured in blue in silico.
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Stem sampling
The research where 3D X-ray μCT scanning was used to
assess xylem fibre PCD (Brereton et al., 2015) used short rotation
coppice (SRC) willow trees (cultivar Resolution-pedigree: [S. viminalis. x (S. viminalis. x S. schwerinii SW930812)] x [S. viminalis. x (S.
viminalis. x S. schwerinii ‘Quest’)] grown in pots for 12 weeks. At week
six the trees were tipped at a 45° angle to the horizontal and held in
place for a further 6 weeks before harvesting (with the stem tied to a
straight bamboo stem every two days to maintain the 45° angle). This
alteration to the vector of gravitational stimulus elicits a unique
change in tissue development known as the reaction wood response
(Andersson-Gunneras et al., 2003; Brereton et al., 2011; Pitre et al.,
2007; Wardrop and Dadswell, 1955).
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Harvest a sample whole stem segment of 6-8 cm from the midpoint of the stem.
Note: This is deliberately chosen in preference to the traditional set
breast height of 1.3 meters in order to allow developmentally comparable
analysis between genotypes of different morphologies.
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Cut two
small triangular notches into the wood 3-4 cm along the segment using a
razor blade (making sure to reach through the bark to the secondary
xylem), both notches should be at asymmetric positions from a transverse
perspective (see Figure 2).

Figure 2. Chlorazol black and Safranin
O staining. Classical sectioning and staining with light microscopy can
be used to help navigate and orientate tissue architecture of the stem
(Panel A). In this context, chlorazol black E (counter-stained with
safranin O) is used to stain the g-layer of g-fibres in tension wood of a
tipped stem sample of SRC willow (cultivar resolution)-this can then be
aligned with images from the μCT scan (Panel B). Note, there is
variation throughout the entirety of the 3D stem (longitudinal variation
in tissue as well as transverse) so multiple notches in the stem
biomass being scanned (one is visible here as a single triangle cut in
panel A) are an important tool to help navigate the 3D volume data from
the scan alongside natural asymmetry of the tissue. Scale bar = 4 mm.
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Cut the segment in half to create two 3-4 cm segments. One 3-4 cm
stem segment (stem A) will be used for X-ray μCT scanning while the
other (stem B) will be used for associated staining and light or
confocal microscopy. The notches will assist in navigation of the
segments (allowing overlay and alignment) and can be removed in silico for 3D µCT render (stem A) or during sectioning (stem B).
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To
make sectioning, staining and X-ray μCT scan differentiation simpler,
bark can be removed at this point from both stem segments by manual
peeling or with a scalpel.
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After harvesting stem segments should
be “fixed” in FAA-formaldehyde, acetic acid, alcohol solution (3.7%
formaldehyde, 5% acetic acid and 47.5% ethanol).
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Drying and pre-μCT scan prep
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After fixation of Stem A for > 3 days, the segment should be
removed from FAA solution and briefly washed in H2O 4 days prior to 3D
X-ray µCT scanning.
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Air-drying of stem A should be performed at
room temperature (no greater than 30 °C) for over 4-5 days in a clean
environment with relatively good air turnover. Forced drying at higher
temperatures can pull H2O from the cell wall (beyond fibre saturation
point) resulting in cell wall collapse. In very dry climates (low
humidity) this can also occur during air-drying.
Note: Air-drying
of stem samples seems an essential step in creating contrast between the
three elements within the biological system under scrutiny: the cell
wall, the protoplast and void space. The important factor of air-dried
samples being that “full” cells, tubular in shape, are easily
distinguished from intracellular void space (which was not the case when
“wet”, as intracellular void space is full of water (Figure 3).

Figure 3. Wet and air-dry biomass 2D computational tomography scan
examples. 3D X-Ray µCT models are rendered from >1,000 2D images,
such as those from a tipped stem sample of SRC willow (cultivar
resolution) shown above. A represents a 2D slice of a sample scanned
“wet” whereas B represents a 2D slice of the same sample after having
been air-dried for 3-4 days. Greater contrast between cell walls, cells
with a protoplast (having not completed programmed cell death) and void
volume is visible in the air-dried sample.
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Once stem A is
air-dried the sample can be secured in a low-density media, usually
standard Oasis® floral foam (a phenolic based polymer, care should be
taken if specifically studying cellulose, as such low densities are hard
to differentiate) and positioned for scan with the structure/s of
interest in the centre of the scan volume.
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Infiltration
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As X-ray absorption increases with atomic number of atoms within a
voxel, stem samples can potentially be vacuum infiltrated with heavy
metals before 3D X-ray µCT scanning. Such as with phosphotungstate
(Staedler et al., 2013).
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Post-μCT scan
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Once stem A
has been scanned (Brereton et al., 2015) a destructive density
measurement can be taken. Basic density (Biermann, 1996; Jourez et al.,
2001) is best suited for biomass density quantification as it
incorporates tissue structural variation often meaningful in terms of
physiology.
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Vacuum infiltrate stem A with water before green
volume is measured via water displacement (Biermann, 1996). Vacuum
infiltration can be performed by pulling a vacuum over submerged wood
samples for around 24 h depending on the material used. Wood oven dry
weight is then measured after drying overnight at 105 °C.
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Calculated as (Biermann, 1996; Jourez et al., 2001): Basic density = oven dry mass/volume.
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Sectioning and staining
Staining of the transverse sections from Stem B allows for different
tissue types to be visualised and quantified within the stem. By clearly
following each stem segment and navigating natural asymmetry (as well
as notches cut into the stems), the in silico 3D X-ray µCT render,
g-layers of g-fibres [chlorazol black E (Brereton et al., 2015; Brereton et al., 2011; Brereton et al., 2012; Robards and Purvis, 1964)],
β-galactosyl Yariv reagent (Tryfona et al., 2012; Kitazawa et al.,
2013), secondary cell wall (safranin O) and remnants of cell contents
(Coomassie and β-galactosyl Yariv) can be aligned and compared in situ.
The in situ β-galactosyl Yariv reagent and Coomassie staining seem to be
without published precedence but are simple and effective. Coomassie
staining is particularly effective for macro navigation (across a whole
transverse section) of cell content remnants using light microscopy, as
classical viability staining can be ineffective in woody tissue (such
as: Tunnel and NBT) due to cell disruption during the sectioning
process. Staining duration and concentrations are only suggestions here
as cell wall structure will vary depending on the plant studied and the
growth conditions.
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Once Stem B is secured in the Reichert sledge
microtome, adjust angle of blade to the correct plane (this may require
some trial and error depending on the material), dampen the sample with
water and cut the thickness of < 30 µm.
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A 48 well plate is useful to perform multiple staining pipelines simultaneously on a common set of sections.
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Using tweezers throughout, first place a section in H2O to hydrate
for 2 min before using section for either chlorazol black E staining,
β-galactosyl Yarive reagent in situ staining or Coomassie in situ staining (cell wall antibodies can also be integrated with analysis
here).
Chlorazol black E staining (example Figure 2)
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Counter-stain by placing sections in a well containing 1% safranin O (aq) for 2-3 min.
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Wash in wells containing H2O x2 for 30 sec.
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Place section in progressive dehydration series: 50% ethanol for 30 sec, 75% ethanol for 30 sec, 100% ethanol x2 for 1 min.
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Place section in well containing 1% chlorazol black E (in methoxyethanol) for 4 min to stain g-layers.
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Wash in well containing 100% ethanol x2 for 1 min.
β-galactosyl Yariv reagent staining (example Figure 4)
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Place section in 0.1 mg/ml dilute β-galactosyl Yariv reagent in 0.15
M NaCl and incubate at room temperature for 4 h on gently shaking
platform.
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Wash as necessary in wells containing H2O.
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Move onto step E9.

Figure 4. In situ β-galactosyl Yariv reagent staining. β-galactosyl
Yariv reagent has long been used to condense arabinogalactan proteins
(AGPs). As the g-layer of g-fibres in tension wood are rich in
Fasciclin-like arabinogalactan protein (Lafarguette et al., 2004;
Andersson-Gunneras et al., 2006), β-galactosyl Yariv reagent works well
as an in situ stain for g-layers in tension wood of SRC willow stem as
well as a protoplast stain (live cells will also include AGPs). Light
microscopy of a tipped stem sample of SRC willow (cultivar resolution)
shows A patterning of tensions wood g-layers and delayed PCD well
aligned those of Figures 2. The same sample magnified using light
microscopy to x63 illustrates: B. Tension wood (TW), C Opposite wood
(OW) and D TW/OW interface show how clear the in situ β-galactosyl Yariv
reagent staining can differentiate these cellular components. Scale bar
= 4 mm.
Coomassie staining (example Figure 5)
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Place section in 1% Coomassie solution and incubate at room temperature for 4 h on gently shaking platform.
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Wash as necessary in wells containing H2O.
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Move onto step E9.
Slide construction
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Rinse staining section in Histoclear clearing agent for 1 min. At
this stage apply DPX to slide to slightly set (1 min or so).
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Place section on to the DPX and apply cover slip, lowering gently. Apply
pressure to force out air bubbles, place some weight (a few grams) on
top and leave to set for 24 h.

Figure 5. In situ Coomassie staining. A. Stitched 25 μm transverse sections stained with 1% Coomassie
solution and counter stained with safranin O. B. Stitched 25 μm
transverse sections without counterstain, remnants of cell contents is
visible if present. While z-stacked images are difficult to assemble
clearly using Coomassie staining, the process was invaluable to assess
remnants of cell content using light microscopy (by eye, as one can move
slightly through 3D space using light microscopes) and led to the
identification of the same PCD patterning using 3D X-ray μCT. Scale bar =
4 mm.
Acknowledgments
The author was financially supported by BioFuelNet Canada. X-ray μCT scanning was performed in collaboration with Farah Ahmed and Dan Sykes at the Natural History Museum with funding from BBSRC Sustainable Bioenergy Centre (BSBEC), working within the BSBEC BioMASS (http://www.bsbec-biomass.org.uk/) Programme. In situ staining techniques were developed in collaboration with Dr. Michael Jasmine Ray.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Brereton, N. J. B. (2016). Sample Preparation for X-ray Micro-computed Tomography of Woody Plant Material and Associated Xylem Visualisation Techniques.
Bio-protocol 6(6): e1767. DOI:
10.21769/BioProtoc.1767.