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Postharvest Biology and Technology 84 (2013) 16–21

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

journa l h om epa ge : www.elsev ier .com/ locate /postharvbio

pplication of optical coherence tomography to non-destructively characteriseind breakdown disorder of ‘Nules Clementine’ mandarins

embe Samukelo Magwazaa,d, Helen D. Fordb, Paul J.R. Cronjec, Umezuruike Linus Oparaa,andra Landahld, Ralph P. Tatamb, Leon A. Terryd,∗

Postharvest Technology Research Laboratory, South African Research Chair in Postharvest Technology, Faculty of AgriSciences, Stellenbosch University,tellenbosch 7602, South AfricaDepartment of Engineering Photonics, School of Engineering, Cranfield University, Bedfordshire MK43 0AL, United KingdomCitrus Research International, Department of Horticultural Science, Stellenbosch University, Stellenbosch 7602, South AfricaPlant Science Laboratory, Cranfield University, Bedfordshire MK43 0AL, United Kingdom

r t i c l e i n f o

rticle history:eceived 18 February 2013ccepted 30 March 2013

eywords:itrusil glands

a b s t r a c t

The feasibility of optical coherence tomography (OCT) for imaging histological changes associated withthe development of a progressive rind breakdown (RBD) disorder of ‘Nules Clementine’ mandarin (Citrusreticulate Blanco.) was investigated. The investigation utilised fruit with different levels of the disorder,carefully selected from a batch of fruit stored for eight weeks at 8 ± 0.5 ◦C. Images of healthy and RBD-affected intact mandarin fruit were acquired using a Thorlabs OCT system based on a broadband 930 nmsource. OCT provided high resolution 2D images of fruit rind to a depth of about 1.1 mm. Immediate

istological characteristicsicrostructural

ruit quality

and non-destructive acquisition of images showing histological and microstructural features in intactrind tissues was demonstrated. The oil glands stayed intact in unaffected fruit and gradually collapsedin RBD affected fruit. At advanced stages of the disorder, the collapsed oil glands became increasinglydeformed and flattened. The study showed that OCT is a promising technique for immediate, real-time andnon-destructive acquisition of images showing histological and microstructural rind features of ‘NulesClementine’ mandarin fruit.

. Introduction

Fresh citrus fruit are prone to various types of postharvesthysiological rind disorders, manifested by a multitude of symp-oms during handling and storage (Magwaza et al., 2012a, 2013). Aostharvest physiological rind disorder of ‘Nules Clementine’ man-arin, commonly referred to as rind breakdown (RBD) is amongeveral commercially important defects affecting the citrus indus-ry (Cronje et al., 2011a,b). RBD is characteristically progressivend starts showing symptoms during storage, about 3–5 weeksostharvest (Cronje et al., 2011b). In export terms, the develop-ent of visible symptoms usually coincides with the commercial

hipping period and/or point of sale. This is therefore extremelyroblematic as rind disorders can lead to large financial losses andustomer complaints (Cronje et al., 2011a).

During early stages, visual symptoms of RBD on ‘Nules Clemen-

ine’ mandarin are manifested as small, irregular, and slightlyunken patches of about 3–6 mm in diameter randomly scatteredbout the flavedo (the outer-most, pigmented part of citrus rind)

∗ Corresponding author. Tel.: +44 (0)7500 766490.E-mail address: l.a.terry@cranfield.ac.uk (L.A. Terry).

925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.postharvbio.2013.03.019

© 2013 Elsevier B.V. All rights reserved.

of the fruit (Cronje et al., 2011a; Magwaza et al., 2013). The sunkenareas associated with RBD occur directly above and among the oilglands of the affected flavedo. The affected areas coalesce, pro-ducing larger lesions, turning reddish-brown to dark-brown, andbecoming dry and necrotic in the severe stages of the disorder withextended storage (Alférez and Burns, 2004; Cronje et al., 2011b).

Currently, the microstructural changes associated with thesedisorders on citrus fruit are evaluated using conventional meth-ods such as light microscopy and transmission electron microscopy(Chikaizumi, 2000; Vitor et al., 2000; Cajuste et al., 2011). Thesemicroscopic techniques can sometimes be expensive, and requirelaborious and specialised destructive sample preparation. Recently,the trend has shifted towards developing reliable and cost effec-tive technologies to non-destructively monitor fruit physiologicaldisorders (Magwaza et al., 2012b). Non-destructive instrument-based methods are preferred because they allow the measurementand analysis of individual fruit, reduce waste and permit repeatedmeasures on the same item over time.

Optical coherence tomography (OCT) is one such non-

invasive and real-time analytical technique currently available toresearchers and is suitable for examining internal structure ofplant tissue. OCT can be used to visualise not only plant tissuesand tissue boundaries but also information on the shapes and

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L.S. Magwaza et al. / Postharvest B

izes of individual cells (Hrebesh et al., 2009; Meglinski et al.,010). OCT technology enables non-destructive and contactlessross-sectional imaging of internal structures in biological objectsroducing 2-D or even 3-D images of plant tissues at a penetrationepth of up to 2 mm from the surface with 5–20 �m resolution

n scattering media such as fruit (Meglinski et al., 2010; Verbovent al., 2013). The aim of this study was to investigate the feasibil-ty of OCT as a non-destructive technique for imaging histologicalhanges associated with the development of RBD on ‘Nules Clemen-ine’ mandarins.

. Materials and methods

.1. Plant material

One hundred and twenty ‘Nules Clementine’ mandarin (Citruseticulate Blanco.) fruit were harvested from 15 uniform markedrees in a commercial orchard at Citrusdal, Western Cape Province,outh Africa (32◦25′ 22′′ South, 19◦ 0′ 53′′ East). Fruit were har-ested on 16 May 2012, at commercial maturity, and receivedll normal postharvest treatments, including fungicides and waxpplication according to industry practices. Fruit were packed inarton boxes and couriered by air at ambient temperature to Cran-eld University in the United Kingdom, arriving within 48 h. Uponrrival, fruit were stored for eight weeks in a temperature con-rolled room at 8 ± 0.5 ◦C, a temperature known to cause the highestncidence of RBD (Magwaza et al., 2013). During cold storage, fruit

ere visually examined and scored at weekly intervals for theevelopment of RBD symptoms over 8 weeks. RBD was scoredy visual inspection on a subjective scale from 0 = no breakdowno 3 = severe breakdown in order to quantify incidence as well aseverity. At week 8, a total of 12 intact mandarin fruit with differentevels of RBD were selected so that they could be examined withhe OCT system. For these evaluations the fruit were sorted intohe four severity levels (0–3) from which three representative fruitere used as replicates.

.2. OCT scanning experimental system

A ‘spectral radar’ OCT system (Fig. 1), using a 930 nm broad-and, near-infra-red super-luminescent diode source (Thorlabs

td., UK), was used for this study. The OCT system was based uponow-coherence interferometry; oscillating signals are produced,he frequency of which corresponds to path-length differencesetween structural discontinuities in the sample and the surface

Fig. 1. Schematic diagram of the OCT

and Technology 84 (2013) 16–21 17

of a reference mirror. The specified resolution of the system was7 �m with maximum accessible optical depth of 1.6 mm in air, butlower in a high refractive-index medium (Ford et al., 2012; Landahlet al., 2012). In the present study, horizontal scan lengths of 1–4 mmwere used, with the horizontal sampling number fixed at a value of1000 horizontally and 512 vertically. The samples were mountedon a translation stage, which was scanned at a constant rate per-pendicular to the image plane. In most cases, only one oil gland wascontained within the imaging area. Initially, the translation stagewas positioned just outside the range containing one end of theoil gland of interest, and was scanned until the other end of thegland just left the imaging area. The physical distance correspond-ing to this scan range was recorded from the micrometre screwgauge attached to the stage. Images were acquired continuously, ata constant rate, during the entire scan period. The scan distance wasdivided by the number of acquired images to obtain the distancestep between images in a particular set.

2.3. Automatic and manual image processing

To achieve automatic image processing, techniques were devel-oped using Matlab® (Mathworks® Inc., USA) to demonstrateappropriate techniques. However, automatic processing was onlyfully successful for images with a clear boundary between the oilgland and surrounding tissue. Therefore, manual processing tech-niques were also developed. A hybrid approach was adopted in thisstudy whereby the perimeters of oil glands were defined manuallyusing a ‘point-and-click’ method to define the path around the edgeof each gland.

2.4. Area and volume uncertainty

A slight peak in intensity was seen approaching the bound-ary, followed by a rapid drop on moving into the gland interior.The drop from peak to near-zero intensity happened within thespace of 4 pixels, or about 9 �m, which was slightly larger than thedepth resolution of the instrument (7 �m), and implied that theboundary could be identified to within about ±2 pixels. In someimages, and particularly at the bottom of deep glands, the uncer-tainty was closer to ±5 pixels. The average error in gland boundaryposition, whether calculated manually or automatically, was esti-

mated at about 10% while the gland dimension exceeded 70 pixels,and decreased rapidly for larger glands. The typical average errorin oil gland areas was estimated to be about 10–20% for ellipsoidalglands, and 20–30% for strongly flattened glands. The error in gland

system used in the experiment.

18 L.S. Magwaza et al. / Postharvest Biology and Technology 84 (2013) 16–21

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ig. 2. OCT images from mandarin rind at four different stages of RBD, with the devere RBD (d). The oil gland of unaffected fruit (a) was almost circular in shape an

ength was estimated to be about 5%, suggesting an uncertainty inolume of 15–25% for ellipsoidal glands, and 25–35% for flattenedlands. Knowing the oil gland area for each image and the step sizeetween successive images, the incremental volume correspondingo each slice was calculated and summation carried out to obtainhe total volume of the gland.

. Results

.1. Visualisation of fruit with different levels of RBD

Image sets were saved for unaffected mandarin samples and athe three stages of progression of the RBD symptom development.ach image, saved as a 1000 × 512 pixel bitmap, was processed toxtract the shape and area of oil glands visible within the image

ig. 3. Automatic image-processing for mandarin rind sample with stage 2 RBD. (a) Raw bunction applied, holes filled and objects labelled. (e) Object size limits imposed, erosion apn original image.

of RBD increasing from 0, unaffected fruit (a); 1, little (b); 2, moderate (c); and 3,ened as the disorder developed. Images are 2 mm wide × 1.1 mm deep.

area. Representative images of oil glands at different stages of RBDare shown in Fig. 2. The oil glands were intact with almost circu-lar shape in unaffected fruit (Fig. 2a) and gradually collapsed inaffected fruit (Fig. 2b–d), depending on the severity of the disorder.At advanced stages of the disorder, the collapsed oil glands becamedeformed and flattened (Fig. 2c and d).

3.2. Image processing

The automatic image analysis procedure was largely successfulfor images similar to that shown in Fig. 3. The image contained

a single oil gland, the perimeter of which was well-matched bythe automatic shape extraction. In results from healthy mandarinsamples (Fig. 4), the oil glands were fully expanded, and signal-to-noise was rather poor in deeper regions of the image. The program

itmap image. (b) ‘Opened’ image. (c) Averaging filter applied. (d) ‘Extended minima’plied to smooth perimeter. (f) Adjust size and extracted gland shape superimposed

L.S. Magwaza et al. / Postharvest Biology and Technology 84 (2013) 16–21 19

Fig. 4. Automatic image-processing for sample with no RBD. (a) Raw bitmap image. (b) ‘Opened’ image. (c) Averaging filter applied. (d) ‘Extended minima’ function appliedbut oil-gland not successfully separated from background.

F mandarin sample with no RBD (above) and a sample with severe RBD (below). All imagesw

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ig. 5. Representative sets of oil-gland perimeters from cross-sectional images for aere taken through the middle section of the oil glands.

as often unable to separate the gland from the backgroundompletely. For consistency, therefore, manual processing wasltimately employed for all images.

The resulting bitmaps for manual processing of images takenhrough the middle section of the oil glands are shown in Fig. 5, forn unaffected sample and a sample severely affected by RBD. Theixel areas within the gland images were calculated automaticallyy the software. From prior knowledge of the physical scanningimensions, it was then straightforward to calculate gland area andolume.

.3. Oil gland volume analysis

Volumes of glands obtained from fruit with different levels ofhe disorder were determined. A histogram was plotted, showing

he volume of each oil gland as a function of the degree of RBDFig. 6). It is plain that the mean volume of the glands decreasesonsiderably as the disorder progresses. The calculated volumesanged from 12 nL in fruit with severe disorder to 150 nL for fruit

Degree of RB D

Fig. 6. Gland volume as a function of degree of progression of RBD from no (0), little(1), moderate (2) an severe RBD (3).

20 L.S. Magwaza et al. / Postharvest Biology and Technology 84 (2013) 16–21

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Fig. 7. 3D representations of oil glands

ithout the disorder. Shrinkage of the oil glands was not homo-eneous. In healthy mandarins, the glands were approximatelyllipsoidal in shape. Most of the volume reduction occurred throughhrinkage in the direction normal to the surface of the fruit, withhe result that the glands became severely flattened in samplesith advanced RBD. The gland surface also became increasingly

rregular as the volume reduced. Re-entrant angles were seen inome of the cross-sections, implying that the tissue had crumpledround the perimeter of the gland. Three dimensional (3D) ren-erings were generated by stacking the bitmaps of the extractedreas. An iso-surface was fitted to the plots in Fig. 7 which gavehe appearance of a solid 3D shape rather than a set of stackedlices.

. Discussion

This study revealed that oil glands remained intact in unaffectedruit and gradually collapsed in affected fruit, depending on theeverity of the disorder. This is similar to observations reportedor postharvest rind pitting on ‘Marsh’ grapefruit, ‘Navelina’ andNavelate’ oranges and ‘Fallgold’ tangerine (Petracek et al., 1998a,b;lférez and Burns, 2004) where oil glands were identified as therimary sites of the damage associated with non-chilling disor-ers. However, the oil glands of ‘Fortune’ (Almela et al., 1992),

Encore’ (Medeira et al., 1999; Vitor et al., 2000) and ‘Clementine’Assimakopoulou et al., 2009) mandarins affected by rind spottingemained intact and unaffected, suggesting that these disordersight not be originating from the oil gland disruption. Therefore,

he disruption of the oil gland during the development of a disordern a certain cultivar of citrus fruit might offer some suggestion abouthe mechanism and the type of disorder. Results of the presenttudy support the hypothesis that oil glands are the primary sitesf damage in RBD and that rupture of these oil bodies could, inurn, release phytotoxic oil into the surrounding cells, causing theollapse of the oil gland and damage to adjacent cellular struc-ures (Petracek et al., 1998b; Alférez and Burns, 2004; Cronje et al.,011a).

Individual cells and cellular structures surrounding the oillands could not be clearly distinguished on OCT images as pre-iously shown on light, scanning and electron micrographs (Knightt al., 2001; Voo et al., 2012). Similarly, in onions (Hrebesh et al.,009) and apples (Verboven et al., 2013), lateral resolution of OCT

mages has also been previously reported to be lower than thosebtained with transmission and confocal microscopy, respectively.rom the two studies, it was concluded that the OCT images wereot as detailed as conventional microscopy, due to the longer

epth of focus for the OCT system. The fundamental limitationf the OCT resolution in the current study was imposed by theource bandwidth, which was about 7 �m. The dense tissues ofntact flavedo and albedo are strongly scattering, resulting in the

a) no, (b) moderate and (c) severe RBD.

attenuation of OCT signal, hence, reduced image contrast at greaterdepth.

Although images from conventional microscopy are known toprovide higher resolution than OCT for identification of rind mor-phological structures (Knight et al., 2001; Cronje et al., 2011a;Voo et al., 2012), it should be noted that both methods have theiradvantages and drawbacks. OCT is a non-destructive technique thatcould open new possibilities for monitoring plant physiology andmorphology, because repeated measurements can be taken duringdevelopment. Furthermore, OCT covers a considerably large fieldof view in both lateral and axial dimensions of the fruit rind, whileproviding similar information to confocal microscopy (Verbovenet al., 2013).

Previous studies have shown that variations in microclimaticconditions during the growing season and within an orchard andeven canopy may influence fruit biochemical profile of the rind andsubsequently play a significant role in fruit susceptibility to RBD(Cronje et al., 2011a,b; Magwaza et al., 2012a). The ability of OCT tonon-destructively visualise oil gland changes associated with RBDalso opens new possibilities to monitor changes in gland structure,gland size, and the number of oil glands per surface area in devel-oping fruit. Understanding gland development and growth rate onrinds of fruit located on different positions of the canopy mightbe useful in understanding why shaded fruit inside the canopy aremore susceptible to RBD than outside fruit (Cronje et al., 2011a;Magwaza et al., 2012a). To further understand the mechanism ofRBD, the next logical step in future studies would be targeting spe-cific spots on fruit with potential of developing the disorder andfollow it over time.

The inability to automatically process images was not unique tohealthy samples. In some RBD affected fruit, there were also diffi-culties in identifying oil gland areas. Cronje et al. (2011a) reportedthat once oil leaks out of the gland it tends to cause so muchdamage to the cells around it that it could become difficult todemarcate the periphery of the oil gland. Therefore, although wesuccessfully demonstrated the potential of OCT in imaging rindmicrostructural features associated with RBD, the sensitivity of thetechnique still needs to be enhanced to increase signal-to-noiseratio, hence improve the image resolution. This could be achievedby using a higher wavelength to reduce scattering, and/or a broaderspectrum. Essentially, the use of different optical parameters andimproved image processing techniques are crucial to improve theperformance of this system.

The oil gland volume observed in this study is in agreement withthat previously reported on grapefruit by Knight et al. (2001) andVoo et al. (2012). These results show there are strong indications

that the gland volume starts to decrease at an early stage duringthe progression of RBD in the fruit, and that more than an order ofmagnitude of change in volume reduction occurs during the courseof the disorder.

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. Conclusion

OCT is a promising technique for immediate, real-time, andon-destructive acquisition of images showing histological andicrostructural rind features of intact ‘Nules Clementine’ mandarin

ruit. OCT provided high resolution 2D images of fruit rind to aepth of about 1.1 mm. The study further demonstrated the powerf image processing procedures to compute volume and 3D modelsf oil glands. RBD was shown to be associated with the progressiveollapse of oil glands and flattening of the flavedo tissue. However,t was observed that the OCT images appeared to have lesser reso-ution compared to microscopic images reported in the literature,ue to the light scattering properties of intact fruit. OCT applica-ion as an imaging technique for plant tissues is currently in itsnfancy and therefore only available to researchers for experiment-ng purposes. It is therefore critical to note that OCT is currentlyot a replacement for light, electron or confocal microscopy, but aotential alternative, in order to perform non-destructive evalua-ion and monitoring of changes associated with RDB and other rindhysiological disorders of citrus fruit.

cknowledgements

This work is based upon research supported by the South Africanesearch Chairs Initiative of the Department of Science and Tech-ology and National Research Foundation. The authors are gratefulo the South African Perishable Products Export Control BoardPPECB) and the South Africa/Flanders Research Cooperation Pro-ramme (Project UID: 73936) for financial support which made itossible to undertake the study. Mr Lembe Magwaza’s study visito Cranfield University was partly funded by the Commonwealthcholarship Commission of the United Kingdom.

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