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Safranine fluorescent staining of wood cell walls

J. Bond a; L. Donaldson a; S. Hill b; K. Hitchcock b
a
Cellwall Biotechnology Centre, b Biomaterials Engineering, Scion, Rotorua, New Zealand
First Published:June2008

To cite this Article Bond, J., Donaldson, L., Hill, S. and Hitchcock, K.(2008)'Safranine fluorescent staining of wood cell walls',Biotechnic

and Histochemistry,83:3,161 171

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Safranine fluorescent staining of wood cell walls

J Bond1, L Donaldson1, S Hill2, K Hitchcock2
1

Cellwall Biotechnology Centre, 2Biomaterials Engineering, Scion, Private Bag 3020, Rotorua, New Zealand

Submitted September 10, 2007; accepted January 28, 2008

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Abstract
Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified
tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow
fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML)
region. We examined the fluorescence behavior of safranine under blue light excitation using a
variety of wood- and fiber-based samples of known composition to interpret the observed color
differentiation of different cell wall types. We also examined the basis for the differences in
fluorescence emission using spectral confocal microscopy to examine lignin-rich and celluloserich cell walls including reaction wood and decayed wood compared to normal wood. Our results
indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression
wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while celluloserich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of
tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission
seems to be due to factors including an emission shift toward red wavelengths combined with dye
quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence
provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.
Key words: cell walls, cellulose, fluorescence, lignin, safranine, wood

Plant cell walls are composed of cellulose, noncellulosic polysaccharides (hemicelluloses and pectins) and frequently lignin. The typical tracheid cell
wall is made up of a primary cell wall and three
secondary cell wall layers (S1, S2, S3), which differ in
the orientation of their cellulose microfibrils
(Donaldson and Xu 2005) and their degree of
lignification (Donaldson 2001, Harris 2006). In all
these layers, the cellulose microfibrils are aligned
within a matrix of lignin and hemicelluloses (Kerr
and Goring 1975). Between each cell is the middle
lamella (ML), which contains lignin and non-cellulosic polysaccharides (Harris 2006). In leaning trees,
a special type of wood called reaction wood forms;
this is known as compression wood in conifers and
tension wood in dicotyledons. In compression wood
tracheids, lignin increases in the outer part of the

Address for correspondence: J Bond, 6A Willow Ar, Hannahs

Bay, Rotorue, New Zealand. E-mail: Suprajac@yahoo.com.
Biological Stain Commission
Biotechnic & Histochemistry 2008, 83(3
4): 161
171.

DOI:10.1080/10520290802373354

secondary cell wall (S2L), but is reduced in the ML

(Donaldson et al. 2004). In tension wood fibers, a
gelatinous, or G-layer, forms as the innermost layer
and consists mostly of cellulose (Bentum et al. 1969,
Joseleau et al. 2004, Pilate et al. 2004, Daniel et al.
2006, Gierlinger and Schwanninger 2006). Fungusdegraded wood also can contain cell walls of
different composition. White rot fungi degrade
lignin and produce partially delignified cell walls,
while brown rot fungi remove the cellulose and leave
a lignin skeleton with little or no polysaccharide
residues (Eriksson et al. 1990). Similar degradation
can occur as a result of weathering mainly due to
exposure to UV radiation in sunlight, which degrades lignin in the cell walls near exposed wood
surfaces (George et al. 2005).
Despite the increasing number of methods for
specifically visualizing compounds within the
wood cell wall, such as antibodies and carbohydrate binding modules (Willats et al. 2000, Hosoo
et al. 2002, Joseleau et al. 2004, Daniel et al. 2006,
McCartney et al. 2005, McCartney et al. 2006), dyes
still are commonly used to screen quickly and
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inexpensively for chemical differences among and

within different cell wall types (Stockert et al. 1984,
Hogetsu 1990, Knebel and Schnepf 1991, Ma et al.
1993, Graham and Joshi 1995, Mori and Bellani
1996, Vazquez-Cooz and Meyer 2002, Li and Reeve
2004, Donaldson and Bond 2005). One of the most
common stains for botanical tissue is safranine
(safranine O) (Ma et al. 1993, Vazquez-Cooz and
Meyer 2002). This dye stains lignin, chromosomes,
nucleoli, cutin, extracts (resins and gums) and cork
(suberin) (Johansen 1940, Kasten 1989, Ruzin 1999,
Horobin 2002). When used for plant material,
safranine typically is used in conjunction with a
counterstain such as fast green (Johansen 1940,
Berlyn and Miksche 1976, OBrien and McCully
1981, Ma et al. 1993) or astra blue (Srebotnik and
Messner 1994, Vazquez-Coos and Meyer 2002), so
that lignified tissue is stained red while unlignified
tissues are stained in green or blue.
Traditionally, plant tissues stained with dyes are
examined using transmitted light; however, many
dyes often are also fluorescent (Haseloff 2003).
Owing to improvements in image resolution and
contrast provided by confocal fluorescence microscopy compared to wide field transmitted light
imaging, superior results can be achieved for a
range of plant tissues, including wood, using this
technique (Knebel and Schnepf 1991, Donaldson
and Lausberg 1998, Hepler and Gunning 1998,
Haseloff 2003, Angeles et al. 2004, Li and Reeve
2004, Donaldson and Bond 2005). Some cell wall
components, such as lignin, autofluoresce with
maximum absorbance in the UV range and require
no dye (Olmstead and Gray 1997, Albinsson et al.
1999, Donaldson et al. 1999, 2004, Donaldson and
Bond 2005).
In general, dyes have specific excitation and
emission wavelengths, but the chemical environment, e.g., pH, may lead to changes in fluorescence
emission. Changes in fluorescence also may occur
owing to increasing dye
dye or dye
substrate
interactions resulting in quenching and/or Fo rster
resonance energy transfer (FRET). Quenching
(Lakowicz 2006) occurs when the fluorescence
intensity of the sample decreases with increasing
dye concentration, as substrate concentration increases, or with the addition of a quenching agent
that binds or associates directly with the fluorophore. Because quenching usually is not advantageous, the optimal dye concentration and staining
time must be found using a dilution series. FRET
occurs when the emission spectrum of one dye
molecule (donor) overlaps with the absorbance
spectrum of another dye molecule (acceptor)
(Periasamy and Day 2005) resulting in loss of
162

fluorescence at shorter wavelengths and increased

fluorescence at longer wavelengths. In some cases,
the donor and acceptor can be the same dye
(Periasamy and Day 2005).
We examine here the fluorescence behavior of
wood cell walls stained with safranine to understand observed color differentiation among cell
wall and tissue types.

Materials and methods

Wood samples and staining
Blocks of Pinus radiata D. Don (radiata pine) normal
and compression wood, Pseudotsuga menziesii
(Mirb.) Franco (Douglas fir), and Populus deltoides
Bartr. ex Marsh. (poplar) tension wood were cut
approximately 1 cm2 blocks and fixed in formalin
acetoalcohol (FAA; 5% formaldehyde, and 5% acetic
acid in 95% ethanol) until they were used. Several
types of fungal-degraded wood also were examined
including brown rot-decayed radiata pine (cellulose
specific fungus species unknown) and two types of
white rot. White rot-degraded samples of radiata
pine were produced by treating wood blocks with a
pure culture of Trametes versicolor (a non-selective
degrader). Samples of Douglas fir degraded by
Phellinus pini (lignin-specific degrader) were collected. Artificially weathered radiata pine was
produced by treatment in a weathering chamber
(Atlas Wi65 weatherometer (Atlas Electric Devices
Co., Chicago, IL)) using a cycle of 102 min of
ultraviolet irradiation followed by 18 min of water
spray for a 3000 h exposure period.
Transverse sections of the wood samples were
prepared at 60 mm thickness using a sledge microtome; the block was kept wet with water during
sectioning. Wood sections and cotton fibers were
stained in 0.1% aqueous safranine (safranin O, CI
50240, #34067 B (BDH, Poole, England)) for
5
10 min, and rinsed three times for 10 min each
in warm water at 30
40oC. Sections then were oven
dried and mounted in immersion oil for fluorescence microscopy. Cotton fibers were prepared as a
whole mount. Sections were examined as quickly
as possible to avoid background staining caused by
safranine leaching from the sections.
A safranine dilution series was prepared using
wood sections stained for 10 min with 0.1, 0.02, 0.005
or 0.002% safranine. Wood sections also were
stained for 10 min with 0.1% safranine, then destained by washing in 50% ethanol for either 1 or 5 h.
To test the effect of pH on safranine fluorescence,
dye was dissolved in water, in water adjusted to

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pH 3 with HCl, or water adjusted to pH 11 with

NaOH. Wood was stained as described above.

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Microscopy
Conventional (filter based) confocal fluorescence
imaging was carried out using a Leica TCS NT
confocal microscope, while spectral confocal imaging was performed using a Leica SP2 spectral
confocal microscope (Leica Microsystems, Wetzlar,
Germany). Sections stained with safranine were
excited at 488 nm and imaged using a BP530 band
pass filter (30 nm band width) and an LP590 long
pass filter (] 590 nm) for conventional confocal
microscopy. Lignin autofluorescence was excited
with 488/568 nm radiation and imaged using a
BP530/LP590 filter set using slow scan speed and
high laser power. To determine whether lignin
autofluorescence contributed to the fluorescence
signal observed when wood was stained with
safranine, we examined a piece of unstained wood
using the same gain settings as used for safranine
imaging. In mature radiata pine, autofluorescence
was nine times weaker than safranine stained tissue
and therefore did not contribute to safranine fluorescence at our settings. All images were collected at
1024 1024 pixels using 4
32 frame averages.
For spectral scanning of normal, compression
and white rot-degraded wood, samples were
stained with 0.1% safranine and excited at
488 nm, and 20 fluorescence images were collected
at 10 nm intervals from 500
700 nm. For the
safranine dilution series, 30 fluorescence images
were collected at 10 nm intervals from 500
700 nm.
Images were 512  512 pixels with four frame
averages. After collecting the images, regions
within the cell wall were selected for spectral
analysis. The ML, S2L, and inner S2 region of white
rot-degraded wood (S2i) were selected to represent
high lignin and low cellulose content. The S2 region
of normal wood and the ML of compression wood
were used to represent low lignin levels. For the
dilution series, spectral analysis was only performed on the S2L region of compression wood
tracheids. For all confocal imaging, we tried to
match the red/green overlay color to that seen
when looking through the eye piece with a green
long pass filter set. This usually was achieved by
saturating the gain on each detector channel for
conventional confocal imaging.

Results
Safranine dye fluorescently labeled the wood cell
wall producing green/yellow fluorescence in the

secondary cell wall and red/orange fluorescence in

the ML region in normal pine and the S2L region of
compression wood (Fig. 1). Our filter sets assign
515
545 nm to the green channel and wavelengths
over 590 nm to the red channel, giving an approximation of the true color of the sample seen when
using a green long pass filter in wide field
fluorescence. The lmax for safranine dye solution
was 588 nm; therefore, the majority of fluorescence
was collected in the red channel. Separation of red
and green emission channels (Fig. 1B,C,E,F)
showed two different intensity profiles: the majority of the green fluorescence was in the secondary
cell wall (Fig. 1B,E), and the red fluorescence was
in the more highly lignified ML (Fig. 1C, arrows),
S3 regions (Fig. 1C) and in the S2L region of
compression wood (Fig. 1F). Lignin autofluorescence of unstained tissue (Fig. 2) showed high
lignin content in the ML, which correlated with
fluorescence seen in the red channel after safranine
staining (Fig. 1C). Lignin autofluorescence was
approximately nine times weaker than safranine
fluorescence, and at the settings used to image
safranine fluorescence, there was no significant
contribution from lignin autofluorescence.

Chemical composition
To determine whether the safranine green/red
fluorescence profiles shown in Fig. 1 are related
to the composition of the cell wall, we examined a
range of cell wall types of varying composition as
shown earlier by chemical analysis (Fengel and
Wegener 1983). Cell wall types that predominantly
fluoresce red, such as poplar ray cells (Fig. 3A),
radiata pine compression wood S2L (Fig. 3B),
brown rot-degraded wood (Fig. 3C), white rotdegraded inner secondary cell wall (Fig. 3D) and
the ML region of tracheids (Fig. 3A,D,E,F and H)
are all high in lignin and/or low in cellulose. The
sensitivity of safranine staining is shown in white
rot-degraded wood (Fig. 3D). This fungus is
reported to be a non-selective cell wall degrader;
however, safranine staining showed fluorescence
differences in the secondary cell wall. Degradation
started from the lumen side of the S2 layer and
showed a deep red band in the inner S2 region (Fig.
3D). At the top of the image, most of the S2 layer
was degraded leaving a thin red residual cell wall
region. Extract found in the resin canals of radiata
pine also showed red fluorescence (Fig. 3H); that is
not surprising given their polyphenolic composition, which is generically similar to lignin (Fengel
and Wegener 1983).
Safranine cell wall staining

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Fig. 1 (Continued)

164

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Fig. 2. Autofluorescence image of radiata pine normal wood. High fluorescence intensity corresponds to high lignin
content. ML, middle lamella. Scale bar40 mm.

Cell wall types that predominantly fluoresce

green, such as the inner secondary wall layer of
poplar tension wood fibers, known as the gelatinous
layer (Fig. 3A), white rot-degraded Douglas fir
(Fig. 3E), resin canal cells (Bamber 1972)
(Fig. 3H), artificially weathered radiata pine wood
(Fig. 3F), and cotton fibers (Fig. 3G), are all low in
lignin and/or high in cellulose. A gradient can be
seen from green to red at the transition from
weathered to normal wood, showing a transition
from low to normal levels of lignin. The secondary
cell walls of poplar (Fig. 3A), radiata pine compression wood (Fig. 3B), non-decayed or partially
decayed regions of brown rot- (Fig. 3C, between
arrows) and white rot-decayed wood (Fig. 3D), nonweathered cell walls (Fig. 3F) and radiata pine
normal wood (Fig. 3H) all have a mixture of lignin
and cellulose and these cell walls appeared varying
shades of yellow.

Quenching
To determine whether quenching affects the fluorescence properties of safranine, radiata pine
compression wood was stained with 0.1%, 0.02%,
0.005% and 0.002% safranine (Fig. 4). This dye

concentration series showed that the red/green

fluorescence pattern that characterizes different
cell wall regions/types became less distinct with
decreasing safranine concentration. At 0.002%
safranine, the color difference between high and
low lignin regions was lost and the cell wall
fluoresced uniformly green/yellow with only intensity differences among cell wall layers (Fig.
4D). Within the S2L region, green fluorescence
increased relative to the rest of the cell wall as
safranine concentration is reduced, suggesting that
quenching occurs within this region at green
wavelengths, but not at red wavelengths where
intensity declined equally in all cell wall regions
with reducing safranine concentration. This is
shown graphically in Fig. 4 (left side) where green
and red lines represent intensity in the green and
red channels. The white lines in Fig. 4 (right side)
show where the intensity profiles for green and
red channels were measured. The intensity profile
for the red and green channels with 0.1% safranine shows the high fluorescence of the S2L in the
red channel compared to the green channel, while
at 0.002% both profiles are the same.
Compression wood also was stained with 0.1%
safranine, then destained for 1 or 5 h and showed

Fig. 1. Confocal fluorescence images of radiata pine normal and compression wood stained with safranine. (A) Normal
wood, red/green channel overlay; (B) Normal wood, green channel fluorescence, highlighting secondary cell walls;
(C) Normal wood, red channel fluorescence, highlighting the ML and S3 layers. Arrows indicate ML and S3 layer;
(D) Compression wood, red/green channel overlay; (E) Compression wood, green channel fluorescence, highlighting inner
secondary walls and ML; (F) Compression wood, red channel fluorescence, highlighting S2L region of the secondary cell
wall. Arrows indicate S2L region. Scale bar40 mm.

Safranine cell wall staining

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Fig. 3. Safranine staining of wood types with varying levels of lignin and polysaccharides. (A) Poplar tension wood. The
gelatinous layer (G) is predominantly made up of cellulose. The ray cell (R) and ML are high in lignin; (B) Radiata pine
compression wood. The S2L region of the secondary cell wall has high lignin content; (C) Brown rot-degraded radiata pine.
Arrows indicate a region of partially degraded wood containing crystalline cellulose, whereas the surrounding cells have
little or no cellulose; (D) Trametes versicolor-degraded radiata pine; non-selective white rot. The inner cell wall (S2i) has
altered chemistry as shown by the deep red fluorescence; the outer S2 (S2o) appears unaffected. The asterisk shows an
undegraded cell; (E) Phellinus pini degraded Douglas fir; white rot, which selectively removes lignin. Arrows show lignindegraded regions of the cell wall; (F) UV-degraded (artificially weathered) wood. Arrows show the exposed region at the
wood surface where lignin has been removed by artificial weathering; (G) Cotton fibers; (H) A resin canal in radiata pine
wood (RC). Resin canal cell walls do not contain lignin and fluoresce green when stained with safranine while extract (E)
fluoresces red. Scale bars40 mm.

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Fig. 4. Radiata pine compression wood stained with (A) 0.1, (B) 0.02, (C) 0.005 and (D) 0.002% safranine. At 0.1%
safranine, green fluorescence quenches in the S2L, as the safranine solution decreases in concentration the S2L becomes
more fluorescent in the green channel. At 0.002%, green and red fluorescence show the same fluorescence profile. The
graphs to the left of each picture represent red (red line) and green (green line) intensity across the corresponding white
line in the image. Scale bar20 mm.

Safranine cell wall staining

167

the same changes in fluorescence as described

above (data not shown). After 1 h destaining,
red/green color differentiation was still present,
but after 5 h, both channels were similar and
resulted in uniform green/yellow fluorescence.
This confirms that quenching is at least partly
responsible for the color differentiation observed
at high safranine concentration.

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Spectral imaging
Confocal fluorescence microscopy showed an approximation of the true fluorescence color of the
sample by assigning colors to the different detector
channels (Fig. 1). We used spectral confocal imaging to determine whether the fluorescence color
differences observed in the different regions of the
cell wall are related to changes in the fluorescence
spectrum of safranine bound to wood.
Wood was stained with 0.1%, 0.02%, 0.005% and
0.002% safranine and the spectrum for each was
measured in the S2L region (Fig. 5). At 0.1%
safranine, the S2L region had a lmax of 581 nm,
which was close to the 588 nm lmax of the 0.1%
safranine solution. The lmax of the S2L cell wall
stained with the three lower safranine concentrations was 567 nm. Although the lmax for the last
three concentrations was the same, the areas
under the curves of the last two dilutions (0.005
and 0.002%) were shifted farther toward the green
end of the spectrum. As dye concentration was
reduced from 0.1 to 0.002%, fluorescence intensity
increased, with the maximum fluorescence intensity occurring at 0.005% safranine concentration
(Fig. 5).
Comparison of fluorescence spectra from regions
within the cell wall in compression wood, normal

Fig. 5. Fluorescence spectra taken from the S2L region of

compression wood. Wood samples were stained with
0.1%, 0.02%, 0.005%, and 0.002% safranine. The safranine staining solution was a 0.1% safranine solution in
water.

168

wood and white rot-degraded wood stained with

0.1% safranine (Fig. 6) showed a shift of lmax to
longer wavelengths (toward the red end of the
spectrum) with increasing concentration of lignin.
In normal radiata pine (Fig. 6A), the lmax of the ML
was 585 nm compared to 575 nm in the S2 region.
In compression wood, the S2L region of the
secondary wall was shifted 10 nm from that in
the S2 region to 585 nm, and the ML was reduced
10 nm to 575 nm, matching the S2 region of normal
wood. The inner cell wall (S2i) of white rotdegraded wood was highly autofluorescent, suggesting a high lignin content, and fluoresced faintly
red with safranine staining (Fig. 3D, arrow) with
the lmax at 595 nm. The outer S2 region (S2o)
appeared similar to undegraded wood.

Fig. 6. Fluorescence spectra of wood stained with 0.1%

safranine. (A) Safranine stained normal wood; (B) compression wood and (C) white rot-degraded radiata pine.
Regions selected were high in lignin and low in polysaccharides (A, ML; B, S2L; C, S2i) or low in lignin and
high in polysaccharides (A, B, S2; C, ML compression
wood).

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Both dye concentration and the amount of lignin

affected the fluorescence spectrum of safranine
stained wood. The fluorescence spectrum in regions of high lignin was shifted toward red
wavelengths and may also have quenched, thus
further reducing the green fluorescence signal.
Regions of low lignin fluoresced more toward the
green end of the spectrum and showed less
quenching.

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Discussion
Safranine staining reveals differences in chemical
composition and can therefore be used as an easy
method for screening samples for the presence of
reaction wood, fungal degradation or variations in
cell wall composition resulting from mutation or
genetic modification. The differential red/green
fluorescence of cell walls with high lignin or high
cellulose eliminates the need to counterstain with
other dyes (Ma et al. 1993, Srebotnik and Messner
1994, Vazquez-Cooz and Meyer 2002), with the
added advantage of improved resolution of confocal fluorescence microscopy over bright field
light microscopy. The counterstains commonly
used with safranine, such as fast green or astra
blue, are not fluorescent, so cannot be used with
safranine for fluorescence microscopy.
Owing to the change in fluorescence emission,
safranine can differentiate high and low levels of
lignin. Regions of high lignin fluoresce red/orange,
while regions with low lignin fluoresce green/
yellow. Quenching, in combination with a shift
toward red wavelengths would explain why the
S2L region of compression wood appears red
compared to the green/yellow appearance of the
adjacent S2 region. This color effect occurs only
when the dye is quenching; therefore, both dye
concentration and staining time must be tested in
advance to give the best effect. The spectral shift to
longer wavelengths when safranine binds lignin
may be the result of FRET (Periasamy and Day
2005), because the prerequisite of overlapping
absorbance and emission spectra and close proximity of dye molecules (Donaldson and Bond 2005)
fits our observations. The nature of safranine
binding to wood has been reported by Stockert
et al. (1984). These investigations report that safranine forms stacked p-p interactions with lignin by
slotting between the aromatic residues. Less is
known about the interaction of safranine with
polysaccharides.

Safranine also stains polysaccharides, as shown

by examination of artificially weathered wood in
which lignin is removed from the surface tracheids
leaving a cellulose residue, and by the G-layer of
tension wood, which contains mainly cellulose.
Safranine interacts in some way with polysaccharides, because if it were unbound within the cell
wall, we would expect it to show the maximum
shift to the red end of the spectrum as shown by
unbound safranine solution (Fig. 5, 0.1% safranine
solution), which is not the case. In general, safranine association with wood is weak and the dye is
readily leached from cell walls by alcohols (Vazquez-Cooz and Meyer 2002). This also precludes
the use of glycerol as a mounting medium. Safranine is a basic dye and thus it is attracted to acidic
sites by polar bonding (Rost 1995).
Fungal-degraded wood is a useful way to characterize safranine fluorescence. The fungi we used
were characterized as selective lignin, polysaccharide, or general cell wall degraders. Using these
samples, we could show all stages of degradation
within a single section. The high sensitivity of
confocal imaging of safranine stained wood was
shown with the white rot fungus Trametes versicolor.
This fungus has been reported to degrade all cell
wall components equally; however, there was a
dark red layer around the inner S2 (S2i) after
safranine staining. This zone was also highly
autofluorescent (data not shown) and therefore
likely to be high in lignin, reflecting an initial loss
of cellulose followed by the collapse of the residual
lignin-rich secondary cell wall.
Pear sclereids have been stained with safranine
and also show some red/green fluorescence differentiation (Angeles et al. 2004). These investigators
used 1% safranine and stained for at least 20 min.
Based on our results, we would expect these
conditions to result in even greater quenching as
shown by the concentration of fluorescence on cell
wall surfaces including the lining of pit canals.
Angeles et al. (2004) reported that the green
fluorescence was more specific to lignin than the
red fluorescence, but this may be due to the
relatively weak lignification of the sclereids. Other
studies reported the lmax of safranine to be
approximately 530
540 nm (Du rrenberger et al.
2001, Horobin 2002) or 590 nm (Kasten 1989)
compared to 588 nm as reported here. Du rrenberger et al. (2001) found that safranine has an
emission maximum of 540 nm when staining starch
grains and primary walls in foods such as yams
and bread. This value agrees with our observations

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of the predominantly green fluorescence of unlignified cell walls. The chemical environment of
safranine may have a large influence on its fluorescence emission characteristics. Haseloff (2003) also
reported different results with safranine fluorescence after embedding in paraffin, where primary
cell walls fluoresced red rather than green. Thus, it
is important to understand the effects of ones
experimental procedures when using this dye.
The color differentiation among cell wall types
shown by safranine fluorescence is due to the
combined effects of quenching and a spectral shift
toward red emission when safranine is associated with lignin-rich cell walls. Reduction of dye
concentration or leaching of dye from the sample
removes this color differentiation. The observed
effects are compatible with the theory that safranine molecules undergo FRET with each other in
areas of heavy staining where the dye molecules
may be in close proximity to each other. The color
differentiation eliminates the need for counterstaining with fast green or astra blue as used for bright
field transmission light microscopy and thus provides a useful method for determining the relative
amounts of lignin and cellulose in wood cell walls.

Acknowledgments
The authors thank Jacqui Ross, Department of
Anatomy with Radiology, Medical and Health
Sciences, University of Auckland, New Zealand
for her help with spectral confocal microscopy; Ian
Hood from Ensis Forest Biosecurity and Protection,
SCION, New Zealand for providing fungal degraded wood; and Tim Strabala from Cellwall
Biotechnology Centre, SCION, New Zealand for
critical review of the manuscript.

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