assemblies of polysaccharides,lipids, and proteins. For example,
arabinoxylans are the main non-starchpolysaccharides that constitute thelongitudinal assemblies in the aleurone
and pericarp cell wall of wheat.
Numerous AFM studies havebeen reported on the structuralcharacterization of the different
biopolymer components of foodproducts or plant materials after they
have been extracted and purified.19-21
One such example is the imaging ofstarch granules extracted before AFM
observation.22-24 Few studies, however,
have been made from whole zones of
the wheat grain in which the biopolymer
structures are kept in theirnatural environment.25-27
This note supplies an overview of how
the latest advances in AFM technology
and techniques provide a better
understanding of the biopolymer
interaction and organization in wheat
grain, starting from intact tissues.
The ease and suitability of combining
fluorescence with AFM is also
demonstrated, highlighting its abiltity toprovide quick identification of the grain
structure, acquire higher resolution
magnification, and clearly elucidate theultrastructure of different wheat
grain tissues.

SAMPLE PREPARATION

High-pressure freezing followed byfreeze substitution has been shown
to provide superior preservation of
the ultrastructure of plant tissues.28
This protocol was used to preparethin sections through wheat grains forinvestigation by AFM and fluorescence.
Small pieces of the peripheral zone of
winter wheat grains (pericarp, testa,nucellus epidermis, aleurone layerand starchy endosperm) were placed
inside a copper platelet and frozen athigh pressure using an EMPACT freezer(Leica-Microsystems) followed by freeze
substitution in acetone/osmium andembedding in London Resin White(LRW), as adapted from Studer et al.29
Semithin (1μm) or ultrathin sections(100nm, 500nm) were cut using a
Microm MT-7000 ultramicrotome andcollected on glass supports for further
staining and/or AFM examination.
Structural staining was conductedwith fluorescent dyes: Nile blue (0.01%in water, excitation at 488nm), whichstains lipid phases; Calcofluor (0.01% in1/1 by vol. ethanol/water, excitation at400nm), which stains cell wall β-glucanbased polysaccharides; Acid fuchsin(0.01% in 1% acetic acid, excitation at453nm), which stains protein phases,
and Acridine orange (0.02% in phosphatebuffer, excitation at 488nm), whichstains metachromatically a varietyof components. Unstained sectionsof wheat tissue also exhibit autofluorescence, a response of cell wall
phenolic compounds.30
As an empirical rule, ultrathin sections
exhibit the lowest fluorescence intensity
but are remarkably flat, whereas
thicker sections have a more significant
roughness with a higher intensityof fluorescence.
The samples were imaged with fully
integrated BioScope II and BioScope
CatalystTM AFMs (Veeco Instruments)
equipped with an AxioObserver (Zeiss).
Most of the images were done in fluid
to preserve the structure of the cuts.
Optical and fluorescent views wereacquired with an AxioCam MRm (Zeiss).
Different cantilevers (Veeco Instruments)
were used to scan samples under varied
conditions (nominal values are given):
OTESPA (k = 2N/m, f0 = 70kHz) for
tapping in air, MLCT
(k = 0.5N/m, f0 = 120kHz) for tapping inliquid, MLCT (k = 0.01N/m) for contact inliquid, and HMX (f0 = 65kHz) and HMXS
(f0 = 30–36kHz) for HarmoniX in air andin liquid, respectively. In addition, forespecially rough specimens with verytall features, topographic images wereacquired using SC-EH cantilevers (TeamNanotec) that have a tip height greaterthan 20μm and a small cone angle (<25°).

Figure 1. Correlation between epifluorescence (A) and AFM height (B) images. 1 = Starchy endosperm; 2 = Starchy endosperm wall; 3 = Sub-aleurone layer; 4 = Starchgranule; 5 = Sub-aleurone wall; 6 = Aleurone cell wall; 7 = Aleurone cell from the aleurone layer; 8 = Aleurone protein body; 9 = Aleurone cell wall between two aleuronecell; 10 = Cellular junction; 11 = Nucellus epidermis; 12 = Tube cells; 13 = Cross cells; 14 = External pericarp. Optical image was acquired using a 10X dry objective. AFM
image (110 x 110 x 5μm) was generated by contact mode in air.

PAGE 2Ultrastructure and Force Property Measurements of Wheat Grain Tissues Using HarmoniX Mode in Fluid

Figure 2. Phase images (150 x 150μm x 100°) (panel A) and (panel B) were acquired by TappingMode in liquid. Several rsp were tested to investigate surface and volume
properties of the wheat cuts. In light tapping conditions (A), the phase signal is more likely to reflect adhesion forces, whereas in moderate/hard tapping conditions (B),
it is more sensitive to viscoelastic behaviors. The optical correlation allowed identifications of the components in the cut, for instance, labeling with calcofluor (panel C)
reveals the β–glucans of the coat (epifluorescence 20X).

All the experiments were carried out
using the same procedures. First
identification of the cell types andcomponents was made using brightfield (BF), digital interference contrast(DIC) and epifluorescence (EF). Thenthe probes were navigated to the area ofinterest for subsequent AFM imaging.
Finally, we made a comparison of opticaland AFM data. Vision® software (VeecoInstruments) was used for off-line
rendering of AFM height data.

and the surrounding resin, whereas
pericarp layers appear with a red-shift
emission.31 The blue square represents
the location of the AFM scan. Panel B
shows the AFM topographic or heightdata as a 3D representation. The easycorrelation between the fluorescent andAFM images using the Catalyst AFM andMIRO allowed rapid identification of thedifferent cell types and components.
This was the key requirement for theremainder of the study.

EASY CORRELATION OF AFM AND
OPTICAL DATATAPPING AND CONTACT MODE ANALYSES

Preliminary experiments were carriedout on five different wheat sections
stained for fluorescent imaging. The
fluorescence image was then used toeasily navigate to a region of interestfor subsequent AFM. All of this wasachieved using Veeco’s new MIRO
(Microscope Imaging Registration andOverlay) software feature, which is
unique to the BioScope II and Catalyst
AFMs. After a quick calibration of thesample and the tip, any optical image
can be imported into NanoScope®
as a background for a whole seriesof AFM experiments. The location ofseveral AFM scans can be selected on
the optical image by drawing a box.
After each scan, each AFM image is
automatically inserted into the opticalimage. One sample is featured infigure 1. When stained with acridine
orange, a metachromatic dye, different
regions of the ultrastructure appearstained with different colors. In panel A,green zones correspond to the cell walls

We then focused on deriving better
insight into the largely undocumented
mechanical properties of the wheatsamples. The phase signal is known
to correspond to surface (adhesionforces) and volume (viscosity, elasticity)properties when scanning parameters
are set properly.32 In phase images, thebrighter signal is interpreted to meanthat more energy is dissipated between
the tip and the sample. Phase contrast
data can be critically dependent upon
the force of tapping. This force can bemodulated by adjusting the ratio setpoint(rsp), which is defined as the ratio
between the amplitude setpoint and thefree amplitude of oscillation. Dependingon this value, we can refer to soft (highrsp) or hard (low rsp) tapping.
Initial tests were carried out using
TappingMode in air to monitor the phasesignal. Under these conditions, thephase contrast was rather weak andpredominately related to topography.
We thus decided to switch to liquid

environment for further analysis. These
data yielded much more relevant results.
The principle of tapping in liquid studies
is summarized in figure 2. If images are
acquired in light tapping conditions (A),where the phase signal is more sensitive
to surface forces, a clear contrast can beobserved between different cell types.
Focusing on the aleurone layer, thephase signal is particularly dark inside
the aleurone cells, including proteins
and lipid bodies (dark granules), whichindicates low adhesion. On the other
hand, the aleurone cell wall with middle
lamellae (the interstitial layer between
aleurone cells) and starch grains appear
much brighter. All other compartments
like the nucellus epidermis or thepericarp exhibit no phase contrast.
If images are acquired using much“harder” tapping conditions (B) wherephase is more sensitive to volume thansurface properties, like elasticity andviscosity, the phase contrast between
aleurone cells and their surrounding
neighborhood is inverted. Furthermore,
it is now much easier to distinguishbetween the different components ofthe pericarp. Once again, immediate
correlation of the fluorescence image
with the AFM image allowed for easy
identification of thedifferent components.
Further experiments were done using
contact mode in liquid, utilizing the“Point & Shoot” features of NanoScope V(see figure 3). A topographic image wasrecorded after the spring constant of theUltrastructure and Force Property Measurements of Wheat Grain Tissues Using HarmoniX Mode in Fluid

PAGE 3

(A). The weakest adhesion forces (B) were found to be focusedin the pericarp (curve 1) and nucellus epidermis (curve 2), thealeurone cells (curve 3) and the starchy endosperm wall (curve4); whereas the aleurone cell wall (curve 5), middle lamella(curve 6) and starch granules (curves 7 and 8) exhibit verystrong adhesion events.
Although the sample preparation used a resin-embedding
step, which may influence the approach-retract curves, theseobservations correlate very well with the phase contrast
observed in figure 2A. All experiments were done using aplain tip but these results certainly open the field of much
more relevant investigations using functionalized tips. We
also compared approach curves to probe the elasticity ofthe sample. A clear contrast could be observed between thedifferent components. The highest Young’s moduli were foundin aleurone cells (but not the protein/lipid bodies), internal
pericarp, and cross cells. Force volume experiments were also
carried out with similar conclusions.
HARMONIX: OPEN ACCESS TO ALL PARAMETERSSIMULTANEOUSLY

Finally, the samples were investigated using HarmoniX mode.
This mode, enabled by the NanoScope V controller, allows
for non-destructive and high-resolution imaging of samples
using forces equivalent to TappingMode, as well as realtime acquisition of data and quantification of various sample
properties, such as stiffness, dissipation, force, etc.
First, the specialty HMX and HMX-S tips (Veeco Instruments)
were calibrated on a well-known polymer sample to achieve
quantitative values. Then the HarmoniX mode was operated inliquid on different types of wheat cuts using both hard and softprobes. The best contrast was obtained using the soft probes,
as shown in figure 4. This wheat cut plane was obtained using adifferent angle of cutting that was more parallel to the aleurone
layer. This explains why the aleurone cells cover 80% of theoptical image. HarmoniX was mainly used in this area, but also
on pericarp areas (not visible here) and starch areas, which arevisible in the bottom left and top right corners of the opticalimage (for clarity, only the AFM images in the aleurone area areshown here).

Figure 3. Point & Shoot experiments on wheat cuts. After capturing a height
image (A: 140 x 85 x 5μm), several spots of interest were targeted, allowingclassification of areas exhibiting low (B) or high (C) adhesion forces.

cantilever was determined. The “Point & Shoot” feature offers
the option to draw a line or a box on such an image, define a
number of points, and then the AFM tip will automatically moveto those locations to capture one approach-retract curve ateach point. For our studies, it was more interesting to choose“manually” a few spots of interest, selected at a variety ofregions on the sample. As explained previously,33 the approach
curve reveals the stiffness/elasticity, whereas the retract curvereflects any adhesion forces between the tip and the sample.
In figure 3, the retract curves are displayed, that is, the curves
associated with adhesion forces, for the eight points of interest
PAGE 4In HarmoniX mode, up to eight channels can be recorded
simultaneously, though only five are shown in figure 4. On
the phase image, the contrast is very similar to what was
shown in figure 2B, which figures since HarmoniX is operated
in “hard” tapping conditions. On the adhesion channel, thelowest adhesion values are recorded inside the aleurone
cells, whereas the surrounding aleurone cell walls aremuch more adhesive, corresponding to the force volumeexperiments (figure 3). Peak force and elasticity values aremuch higher inside the aleurone cells (except inside protein
and lipid inclusions, which are known to be very soft) than in
the surrounding aleurone cell walls. This again agrees withthe previous force volume experiments. Finally, much moreenergy is lost in the cells than in the cell walls. As the tip wascalibrated, the mean Young’s moduli could be extracted fromthe stiffness channel using the bearing analysis function of theUltrastructure and Force Property Measurements of Wheat Grain Tissues Using HarmoniX Mode in Fluid

Figure 4. HarmoniX imaging in liquid environment from an ultrathin section of a resin-embedded wheat aleurone tissue. The epifluorescence image shows the majorpart of the cut consists in aleurone cells surrounded by starchy zones seen in bottom left and top right corners of the image. The yellow frame represents the AFM
scan location where HarmoniX measurement was performed after calibration of the tip on a polymer sample. Five of the eight AFM channels that can be recorded
simultaneously are shown on this image; phase (z-scale = 30°), adhesion (40nm), peak force (200nm), dissipation (arbitrary units) and stiffness (z-scale = 5GPa). The
information displayed on these five channels is in total agreement with both the data found in literature and the results of the experiments carried out on similar samples
in regular TappingMode and force mode (see figures 2 and 3).

NanoScope V. Example values for thecalibration were 4.6GPa for the aleurone
cells, and 2.4GPa for the surrounding
aleurone walls.
Similar comparisons and calculations
were achieved with other areas (e.g.,pericarp and starchy endosperm
zones), and confirmed the relevance ofcomparing the results of different
AFM modes.
CONCLUSION

The combination of AFM andfluorescence imaging using theBioScope II and Catalyst AFMs
provided useful insight into theultrastructure of intact wheat graintissues. These fully integrated tools
allowed the simultaneous recordingof both fluorescent staining of specificcomponents and AFM signals. The
unique potential of HarmoniX mode,
enabled by the NanoScope V controller,
was also shown to be very useful toprobe the surface and volume propertiesof a plant tissue. Working in fluid
environment was essential to allow
preserving the structure of the cut andwould not have been possible on a nativesample. The next obvious step is to map
more precisely the different HarmoniX
data as a function of the cell layers
and components of a variety of resinembedded or native grain samples. This
new emerging technology opens a wide
range of applications in food researchand plant tissue investigation.

Ultrastructure and Force Property Measurements of Wheat Grain Tissues Using HarmoniX Mode in FluidPAGE 5