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 in
530   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 fewer
NMR 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. 12
534     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 R
NMR micro-imaging of raspberry                                                                                          535

imaging, as in NMR spectroscopy, the dynamic                        HENDERSON, C .  (1989). Aminoalkylsilane: an inexpensive simple
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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
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