Nuclear magnetic resonance (NMR) micro imaging of raspberry fruit: further studies on the origin of the image
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Neiv Phytol. (1992), 122, 529-535
Nuclear magnetic resonance (NMR) micro
imaging of raspberry fruit: further studies
on the origin of the image
BY B. A. G O O D M A N \ B. WILLIAMSON^ AND J. A. CHUDEK^
^ Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
'^Department of Chemistry, University of Dundee, Dundee DDl 4HN, UK
{Received 24 February 1992; accepted 1 July 1992)
SUMMARY
Nuclear magnetic resonance (NMR) micro-iniages of mature red raspberry (Rubus idaeus L.) fruits and their
separated components have been produced under a variety of experimental conditions with the objective of
understanding factors which determine image quality in different types of tissue. By varying the delay time
between image accumulation sequences it has been shown that water molecules in drupelet surface layers in
contact with other drupelets, and in the vascular bundles and traces in the receptacle and drupelets have shorter
relaxation times than those in other tissues of the fruit. By performing separate T^- and T,-weighted imaging
sequences it was shown that the short relaxation times in the vascular tissue was primarily a T^ effect. This is
probably caused by paramagnetic ions because electron paramagnetic resonance (EPR) spectroscopy showed
significant amounts of the manganese(ii) ion in a spectrum of an aqueous extract of receptacle tissue. The cause
of the short relaxation times in the drupelet surface layers is less clear, but we suggest that epicuticular waxes or
the suberized cuticular layer of the epidermis might be responsible.
The appearance of entire seeds, including the mature embryos, as featureless black regions in the images has
been shown to be due to the lower concentration of mobile protons in these structures compared with that in the
surrounding mesocarp, and not to differences in relaxation properties of the protons. Consequently, internal detail
of the seeds could only be obtained by separating them from the fruit.
Key words: NMR micro-imaging, relaxation effects, paramagnetic ions, EPR spectroscopy, raspberry.
paramagnetic niolecules sensitively and specifically
INTRODUCTION
(e.g. Goodman & Raynor, 1970) and has been used
In a recent publication (Williamson, Goodman & previously in the study of free radicals (e.g. Good-
Chudek, 1992) we described the use of NMR man, McPhail & Linehan, 1986; Hepburn et al.,
microimaging to follow, non-invasively, some of the 1986) and transition metal ions (e.g. Goodman &
structural changes that occurred during the ripening Linehan, 1979; McPhail, Linehan & Goodman,
of red raspberry fruits. In that work a comparison 1982) in plant tissues. However, the similar be-
was made of the image quality that was produced by haviour of the images of water in the vascular supply
the pulsed gradient spin-echo and gradient-echo to the seeds within drupelets and the connecting
procedures, with the objective of optimizing the surface regions of the drupelets could not be
resolution of features of histological interest. investigated by this technique. We have also
In the present paper we have investigated the examined possible explanations for the apparent
causes of some enigmatic features of the images absence of internal structure in the N M R images of
derived from that work. By varying the delay times seeds within intact fruit by comparing results
between image accumulation sequences, the relax- obtained . from whole fruits, separated single
ation properties of water in the difTerent types of drupelets and isolated seeds.
tissue have been investigated, and the possibility that
the short relaxation times observed for the vascular
MATERIALS AND METHODS
tissues in the receptacle are the consequence of
Fruit
endogenous paramagnetic ions has been investigated
by electron paramagnetic resonance (EPR) spec- Fruits of the primocane-fruiting red raspberry
troscopy. This technique is able to characterize {Rubus idaeus L.) cv. Autumn Bliss were produced in530 B. A. Goodman, B. Williamson and y. A. Chudek Figures 1-8. Spin-echo NMR images of raspberry
NMR micro-imaging of raspberry 531
a glasshouse and harvested at the red ripe stage of mortar and crushed to a fine powder. This powder
ripeness with the receptacle still attached. was suspended in c. 30 ml ultrapure HgO and
centrifuged at 15 0 0 0 ^ for 10 min. The clear super-
natant was decanted, reduced to a volume of c. 1 ml
NMR micro-imaging in a rotary evaporator at 40 °C and stored at 4 °C
prior to analysis. An EPR spectrum of this solution
N M R imaging of an intact fruit was carried out at was obtained at ambient temperature on a Bruker
20 °C in high humidity using the procedure de- FSP 300F spectrometer operating at X-band fre-
scribed by Williamson et al. (1992). A receptacle quencies in order to investigate the possible presence
detached from the drupelets 1 d after harvest was of paramagnetic ions.
also imaged in order to provide detailed data on the
arrangement of the vascular tissues. Some drupelets
were also separated from the fruit and examined Conventional histological studies
individually because they could be imaged with
greater resolution than the entire compound fruit. Other receptacles were fixed in 2-5 % glutaraldehyde
Some seeds were separated from the mesocarp using in 0-1 M phosphate buffer at pH 7-0, dehydrated
a scalpel, cleaned by blotting on filter paper, and through an ethanol series and embedded in L. R.
stored fresh for up to 48 h before examination. White resin (TAAB Laboratories Equipment Ltd,
All images were acquired using a Bruker Aldermaston, Reading, Berks., UK). Semi-thin
microimaging unit fitted with a 25 mm coil attached sections (3 fim thick) were cut dry using a Reichert-
to a Bruker AM 300/WB Fourier Transform NMR Jung microtome (Model 1140/Autocut) with 12 mm
spectrometer (7-1 T ; ^H, 300 MHz). Data were glass knives and sections were mounted on micro-
collected using a standard spin-echo pulse sequence scope slides coated with 3-aminopropyltriethoxy-
as described in Williamson et al. (1992). For each silane (Henderson, 1989). Sections were stained by
image the transmitter power was attenuated to give toluidine blue at pH 4-4 (O'Brien & McCully, 1981)
90° and 180° Hermite soft pulses of 4000 fis length. and examined by bright field microscopy.
Delay times between pulses were varied and the Whole fruits were also sectioned at c. 50 fiva using
relevant details are given in the captions to the a Jung large freezing microtome, in which the
figures. An echo time (TF) of 18 ms was used for all specimen was frozen by means of a jet of liquid COg
of these accumulations. Inversion recovery T^- and while supported in a matrix of Holt's hypertonic
spin echo T2-weighted images (O-0001-lO-O s) were gum-sucrose medium (0-88 M sucrose containing
also recorded using the snapshot FLASH imaging 1 % gum acacia). Sections were stained and mounted
sequences of Haase (1990). All images were in 0-1% aniline blue in 0-1 M K3PO4.HgO and
accumulated into 256x512 word matrices which examined by fluorescence microscopy (Williamson,
transformed into 256 x 256 pixel images. Voxel McNicol & Dolan, 1987).
dimensions, which are also given in the captions to
the relevant figures, were obtained by appropriate
setting of the spectrometer acquisition parameters. RESULTS
Effects of variations in acquisition sequence delay
times on NMR images of whole fruit
EPR spectroscopy The influence of pulse delay time on the N M R
An aqueous extract of receptacle tissue was prepared images of median transverse and longitudinal slices
by the following procedure using glassware that had of the same red ripe raspberry are shown in Figures
been rigorously washed with 1 M HCl and ultrapure 1-^8. The major changes that were seen with
HgO (NANOpure II, Fison's Instruments, Crawley, progressive shortening of the delay times between
Sussex, UK). Thirty fruits were excised from pedicel image accumulations involved the surface layers of
and calyx using a scalpel and the receptacles were drupelets where they were tightly adhering to
then removed manually, frozen in liquid Ng in a adjacent drupelets, and the vascular bundles in the
Figures 1-4. Spin-echo NMR images of median transverse slice through a red raspberry fruit wdth acquisition
delay times of 10 s (Fig. 1), 2 s (Fig. 2), 04 s (Fig. 3), and 0-08 s (Fig. 4), showing single drupelets (D), each
with an elliptical black seed. The drupelets are arranged tightly in a ring around the central receptacle (R). Note
the increasing brightness of vascular bundles (B) and of surfaces between drupelets with decreasing delaj^ times.
Each image, which has voxel dimensions of 60 X 60 x 500/tm, represents the summation of 4, 4, 8 and 16
accumulations respectively.
Figures 5-8. Spin-echo NMR images of median longitudinal slice through a red ripe raspberry fruit with
acquisition delay times of 10 s (Fig. 5), 2 s (Fig. 6), 04 s (Fig. 7), and 0-08 s (Fig. 8). AH other experimental
parameters were the same as for Figure 1.532 B. A. Goodman, B. Williamson andy. A. Chudek
receptacle. Each of these features showed a pro-
gressive brightening with decreasing delay times,
indicating that the water in these regions has much
shorter relaxation times than in the other parts of the
specimen.
The image of the vascular supply to the seeds
VI
appeared dark in the images taken with the longest
delay times (e.g. Figs 1, 5). With decreasing delay
CO
times the contrast with the surrounding mesocarp
tissue decreased and the two types of tissue became
virtually indistinguishable at the shortest delay times
w
(e.g. Figs 4, 8). Thus the water in these vascular
elements also has shorter relaxation times than that
in the surrounding tissue, but in this case the water
contents of the vascular elements are considerably
lower.
320 340 360 380
T^ and T^-weighted images of whole fruit Magnetic field (mT)
Inversion recovery T^-weighted images of whole Figure 9. First derivative X-band EPR spectrum of
fruit using the snapshot FLASH imaging sequence aqueous extract of receptacle of red raspberry at ambient
temperature. Microwave frequency, 9-8573 GHz; micro-
of Haase (1990) produced results that were es- wave power, 60 mW; modulation frequency, 100 kHz;
sentially similar to those reported above for modulation amplitude, 0-8108 mT; time constant,
variations in delay times. With long T^ (10 s) the 81-92 ms. The characteristic spectral parameters are de-
intensities of the protons in the vascular bundles in termined as follows: (i) the hyperfine coupling constant,
the receptacle were significantly lower than in the A, is equal to the mean separation between adjacent
mesocarp of the drupelets, but w'ith decreasing component peaks (i.e. points where the first derivative
curves cross the baseline), and (ii) the spectroscopic
values of T.^ (from 10-0 to 0-05 s) there was a splitting factor, g, is given by 71-44775 v/B, where v is the
progressive reduction in the brightness of the images microwave frequency in GHz and B is the magnetic field
of the drupelets, whereas the vascular bundles in the in mT at the centre of the spectrum.
receptacle remained essentially unchanged. No
further changes occurred between values of 0-05 and
0-0001 s.
The spin echo Tg-weighted images did not exhibit
any signiflcant differential response of the different
types of tissue to variations in Tg. In the range
lO'O—0"0001 s there was simply a progressive decrease
in overall image intensity with decreasing time.
EPR speetroscopy of water extract of receptacle
To investigate the possibility that short relaxation
times for the water in the vascular elements of the
receptacle were the result of interaction with en-
dogenous paramagnetic ions, an EPR spectrum was
obtained of a w^ater extract of receptacle tissue (Fig.
9). It consists of two distinct components; a major
6-peak resonance with parameters g = 2-0 and
A = 9"4 m T , along wath a weaker single peak Figure 10. Spin-echo NMR image of median transverse
resonance wdth g = 2-0. slice through an isolated receptacle of a red ripe raspberry
fruit showing traces (T) for drupelets departing from the
receptacular stele and delineation of xylem (X) and phloem
NMR determination of the vascular architecture of (P). The image represents the sum of eight sets of
the receptacle accumulations. A delay time of 2 s between image
acquisitions was used and voxel dimensions are
Sixteen separate slices were imaged through a 25 X 25 X 200
receptacle from which drupelets had been removed
in order to determine the arrangement of the vascular cylinder ran along the main axis of the receptacle,
elements. A typical transverse image from the with vascular traces to each drupelet departing from
median slice is shown in Figure 10. The vascular the cylinder in a spiral pattern leaving fewerNMR micro-imaging of raspberry 533
Figure 12. Spin-echo NMR images of a single drupelet of
a red ripe raspberry fruit taken with acquisition delay time
of 10 s showing a central seed (black) with serrated edge
'cut' in longitudinal section. The image represents the
summation of eight sets of accumulations and has voxel
dimensions of 25 x 25 x 200 /tm. Insert: Spin-echo NMR
projection image of an isolated seed of a red ripe raspberry
fruit taken with acquisition delay time of 2 s. The image is
the summation of 32 accumulations with each pixel
representing 25 x 25 /^m projected areas.
within each surface depression. The receptacular
stele comprised a series of separate bundles and a
single vascular trace departed from the stele to
supply every drupelet previously attached in a spiral
arrangement (Fig. 11). Therefore, in serial slices the
number of bundles visible in the stele was greatest in
proximal slices and decreased towards the distal apex
of the conical receptacle as fewer drupelets remained
to be supplied with water and nutrients.
Examination of frozen sections of mature fruits by
fluorescence miicroscopy showed that where two
drupelets were pressed together the cuticles of the
two opposing epidermal layers were in intimate
contact or fused. Epidermal hairs were generally
absent from the middle of the flattened radial facet of
the drupelets, but at the junctions between three
tightly packed drupelets a triangular air space and a
dense tangled mass of trichome hairs were present.
Figure 11. Portion of raspberry receptacle in transverse
section seen b^^ bright field light microscopy showing NMR imaging of seeds
arrangement of xylem (X) and phloem (P) in vascular
bundles (B) and departure of traces (T) to previous points An N M R image of a single drupelet is shown in
of attachment of drupelets (D) ( x 80). Figure 12 with higher resolution than could be
achieved with measurements on an intact compound
'bundles' in distal slices than in proximal ones. Gaps fruit. The edge of the seed appears serrated, but
in the cylinder above departing traces left the there was no evidence of any internal structure from
receptacular stele as a series of separate bundles with the embryo, or indeed of any mobile protons for
clear demarcation of inner xylem and outer phloem. images acquired with a range of different delay times
(0-4—10 s). With an isolated seed a very weak image
(not shown) was observed on a 200 [im thick slice.
Conventional histology of mature receptacle However, by using a N M R pulse sequence that
The receptacle in transverse sections had a crenulate produces a projection of the mobile proton density
surface with an attachment point for a drupelet through the whole specimen, an image (Fig. 12534 B. A. Goodman, B. Williamson and J. A. Chudek
insert) was produced which shows clearly the surfaces. External dome-shaped drupelet surfaces
presence of mobile protons along with evidence for are covered with a mass of epidermal hairs. At the
some poorly defined internal structure. edges of the areas of contact between drupelets,
bands of twisted masses of hairs extend from the
surface just above the vascular connection to the area
DISCUSSION where the drupelet domes diverge, but hairs are
Variations in water relaxation rates ^^^^^^ ^^^^ ^^^ ^^^^^^^ ^^^^ ^f contact with neigh-
If during the time between the application of each bouring drupelets (Robbins, Sjulin & Rasmussen,
image accumulation sequence the individual protons 1988). This structure is supported by our present
were able to relax completely back to their equi- observations and the waxy adhesion between
librium conditions, then the intensity of each pixel in drupelets described by Reeve, Wolford & Nimmo
the image would represent the total amount of (1965) can be attributed to the epicuticular waxes
mobile protons in the corresponding volume of the and cutin of two opposing epidermal layers,
specimen. However, there are variations between all It would appear that the regions with rapidly
of the images in Figures 1-8 indicating that relax- relaxing protons in the N M R images are those where
ation of protons after each radio-frequency pulse was hairs are absent from the drupelet surfaces; the hair-
incomplete in all of the images acquired with covered external surfaces and regions where three
accumulation delay times of 2 s or less. The image drupelets join together show no such enhancement
shown in Figure 2 is essentially equivalent to that in relaxation rates. Indeed, the latter regions appear
presented in Figure 13 of Williamson et al. (1992), dark in the N M R images, presumably because of the
where experimental conditions were selected for presence of air spaces. The waxy adhesion reported
optimum speed of image accumulation. Conse- by Reeve e? a/. (1965) may be directly responsible for
quently, the intensities of the voxels (defined the short relaxation times of the protons in these
volumes within sample) in the images presented by regions of the fruit since it has been observed in this
Williamson et al. (1992) do not simply equate to laboratory that waxy substances are able to enhance
mobile proton contents in the corresponding volume the brightness of N M R images from water molecules
elements of specimen. in contact with them (Chudek, unpublished results).
EPR measurements of a water extract of
receptacles show the presence of significant amounts ^^ .^^^ .^ ^^^ ^^^^^^^ ^^^^^
of paramagnetic ions. The parameters for the
principal resonance in the EPR spectrum (Fig. 9) are In all of the images of intact fruit both in the present
characteristic of the solvated manganese(ii) ion work and that reported by Williamson et al. (1992)
(Goodman & Raynor, 1970), whilst the minor the seeds appeared as featureless black regions. No
component probably corresponds to a free radical evidence for mobile protons was seen with either
species. Therefore, the enhanced relaxation rates of wide variations in accumulation delay times (Figs
the protons of water in the vascular elements of the 1-4, 5-8) or T^- or T^-weighted images, even though
receptacle were probably caused by the presence of other investigations on a range of seed types have
paramagnetic ions in solution, and predominantly revealed internal structure and, on occasions, sep-
the manganese(n) species. This is a well-known arate images from water and lipid components
phenomenon; the addition of trace quantities of (Sarafis et al., 1990; Goodman & Chudek, un-
paramagnetic molecules to samples has long been published results). High resolution images of
used as a method to shorten relaxation times for separated drupelets also failed to reveal any internal
NMR spectroscopy and to provide contrast in features in seeds. The serrated external structure of
magnetic resonance imaging. the endocarp was due to the presence of undulating
We postulate that a similar process occurs in the and overlapping layers of sclereids (Reeve, 1954).
vascular supply to the seeds, although that has not Very weak images were obtained, however, when
been proven in these measurements. Also the results seeds were isolated from the surrounding mesocarp,
show that the water contents of these vascular but definitive evidence for mobile protons was only
elements are considerably lower than in the sur- obtained when we used an imaging sequence that
rounding mesocarp. This is presumably because of produced a projection through the entire specimen,
the low water content of the comparatively thick Short T.^ times have been cited (Connelly et al.,
walls of the vascular elements, and may reflect the 1987; Morris et al., 1990) to explain the low
change in water status following plugging of vessels intensities often found in images of seeds. However,
after fruit abscission (MacKenzie, 1979). in our results it is likely that the blackness of the
It is not possible to determine whether or not the seeds in intact fruit and within isolated drupelets is
same process is responsible for the enhanced relax- the result of a combination of small size and
ation rates of the water in the connecting surface comparatively low mobile proton density compared
regions of the drupelets, or whether in this case it is with that in the surrounding mesocarp, and not as a
caused by some other property of the drupelet result of different relaxation properties. In N M RNMR micro-imaging of raspberry 535
imaging, as in NMR spectroscopy, the dynamic HENDERSON, C . (1989). Aminoalkylsilane: an inexpensive simple
preparation for slide adhesion. The Journal of Histotechnology
range of the data is finite and often results in 12, 123-124.
information loss - in this case the intensity of the HEPBURN, H . A., GOODMAN, B . A., MCPHAIL, D . B., MATTHEWS,
protons in the seeds. S. & POWELL, A. A. (1986). An evaluation of EPR
measurements of the organic free radical content of individual
seeds in the non-destructive testing of seed viability. Journal of
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MACKENZIE, K. A. D. (1979). The structure of the fruit of the red
ACKNOWLEDGEMENTS raspberry {Rubus idaeus L.) in relation to abscission. Annals of
Botany 43, 355-362.
We thank Dr P. Such of Bruker Analytische Messtechnik MCPHAIL, D . B., LINEHAN, D . J. & GOODMAN, B . A. (1982). An
GmbH for producing the EPR spectrum and acknowledge electron paramagnetic resonance (EPR) study of the uptake of
the Scottish Office Agriculture and Fisheries Department vanadyl by wheat plants. New Phytologist 91, 615-620.
for funding this work. MORRIS, P. G., DARCEUIL, H . E., JASINSKI, A., JHA, A. K.,
MCINTYRE, D . J . O . & NoRTHCOTE, D. H. (1990). NMR
microscopy of the germinating castor bean. Philosophical
Transactions of the Royal Society London A333, 487-493.
O'BRIEN, T . P. & MCCULLY, M . E . (1981). The Study of Plant
Structure, Principles and Selected Methods. Termarcarphi Pty.
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