PII: SO300-5712(96)00081-4 Journal of Dentistry, Vol. 26,No. 2, pp.

89-96,1998
0 1998ElsevierScience
Ltd. All rightsreserved
Printedin GreatBritain
0300-5712198 $19.00+0.00
ELSEVIER

A comparison of two histological validating

techniques for occlusal caries
D. N. J. Ricketts, T. F. Watson, P. J. Liepins and E. A. M. Kidd
Department of Conservative Dentistry, United Medical and Dental Schools, Guys Hospital, London, UK

ABSTRACT
Validation of a diagnostictechniqueis important to establishwhether it actually measureswhat it is
purported to measure.However, the accuracy of the validation techniqueper se can influence the
apparentaccuracy of the diagnostictechnique.
Objectives: The aim of this study was to describetwo alternative histologicalvalidating techniquesfor
occlusalcariesand to compare quantitative depth measurements of carious lesionstaken using each
method.
Methods: Thirty sections(meanthickness0.67mm) were cut to include two to four discretesitesin 10
freshly extracted teeth. The first histologicalvalidating techniqueuseda microfocal X-ray unit to produce
magnifiedhigh definition radiographicimagesof the sectionsor macroradiographs.An imageanalysis
systemwas used to make quantitative measurementsof the lesions(if present) with respect to the
enamel-dentinejunction (EDJ). The secondvalidating techniqueuseda confocal microscopeto image
beneaththe cut surface of the section. Quantitative measurements were taken from the fluorescence
imagesof both sidesof each sectionand a meandepth measurementcalculated.
Results: Complete agreementwas found between the two validating techniquesfor the subjective
interpretation of the presenceand extent of caries.A strongpositive relationshipwasfound betweenthe
two histological validating techniquesfor depth measurements made of dentine cariesfrom the EDJ
(~0.93, P<O.OOl).Depth measurements made from the macroradiographswere greater than from the
confocal fluorescenceimages(meandifference=0.41mm).
Conclusions: Both validating techniquesenablethe identification of soundsites,thosewith enamelcaries
and dentine caries. However, quantitative assessments made with each technique could result in
disagreement.0 1998Elsevier ScienceLtd. All rights reserved

KEY WORDS: Histology, Validation, Microfocal radiography, Confocal microscopy, Occlusal caries

J. Dent 1998; 26: 89-96 (Received 2 April 1996; accepted 30 October 1996)

INTRODUCTION The marked decline in dental caries seen in todavs<

population has been accompanied by a change in
When a new diagnostic system is introduced, it is distribution of tooth surfaces affected. Occlusal caries
essential to validate decisions, readings or measure- now accounts for the majority of carious lesions and as
ments. That is, it is important to establish whether it such has received much attentionlP6. Because of the
actually measureswhat it is purported to measure. This
reported difficulty with which occlusal caries is diag-
is true in dentistry and in particular in validating
nosed and monitored, a number of new diagnostic
diagnostic decisions made for the presence or absence
techniques have been described and investigated7. It is
of dental caries.
important that such studies are initially carried out in
the laboratory so that accurate validation and quanti-
Correspondence should be addressed to: Dr D. Ricketts, Depatt-
ment of Conservative Dentistry, United Medical and Dental
fication can be made on sectioned teeth. In a clinical
Schools, Floor 25 Guys Tower, Guys Hospital, London Bridge, environment only the true positive decisions could be
London SE1 9RT, UK. Tel.: 0171 955 4937/4045. Fax: 0171 95.5 ethically validated, as it would be unacceptable to cut a
4935. cavity in a tooth thought to be sound.
90 J. Dent. 1998; 26: No. 2
It has been recognised, however, that the apparent
accuracy of a diagnostic technique can be influenced by
the method of validation and the person carrying out
the validation8. Whilst great emphasis has been placed
upon comparisons of diagnostic techniques, little atten-
tion has been paid to the differences between validation
techniques, making comparisons between studies diffi-
cult. Studies which have compared histological tech-
niques have mainly concentrated on approximal lesions.
With the exception of a study by Hintze et al. few
studies to date have compared validation techniuqes for
occlusal cariesg. In this study, stereomicroscopy, film
radiography, microradiography and naked-eye inspec-
tion of tooth sections were compared. Stereo-
micropscopy was found to be the most trustworthy for
the identification of sound and carious teeth.
Classically, polarised light microscopy and micro-
radiography have been described for the histological
validation of early demineralisation. However, for
these techniques sections have to be ground to about
1OOym. Where occlusal caries is concerned, initial
enamel lesions may have multiple foci within such
sections and may develop at independent sites along a
single fissure system1213. As the lesions enlarge they
may coalesce into a single larger lesion. Polarised light
microscopy and microradiography therefore have a
number of disadvantages for the validation of occlusal
caries. Firstly, it is time consuming as the sections are
initially cut with a saw and then gradually ground
down. Secondly, the deepest aspect of the lesion may be
missed or lost in the grinding debris and finally, such
thin sections are readily damaged: especially if there is
substantial demineralisation present.
Other validation techniques have been described for
laboratory studies including simple hemisection of
teeth1415, viewing thicker sections (approximately
700-1000 pm thick) under a stereomicroscope6 and
examination of conventional radiographic images of
such sections17. The use of thicker sections overcomes
the problems of polarised light microscopy and
microradiography.
This study aims to describe and compare the results
obtained from two histological techniques, adapted for
the identification and quantification of occlusal caries,
using thick sections. The two histological techniques
investigated were microfocal radiography (not to be
confused with conventional film radiography or
microradiography) and confocal microscopy.
MATERIALS AND METHODS
Tooth selection
Ten freshly extracted third molar teeth from 10 young
adults were collected, cleaned and stored in saline to
which a few crystals of thymol were added to inhibit
further bacterial growth. The teeth were fully erupted,
non-cavitated and had been present in the mouth for
over 2 years. Two to four discrete sites within the pit
and fissure system of each tooth were chosen for
investigation (total 30 sites) and the clean dried surface
site recorded as:
Sound;
White spot or brown spot at the entrance to the
fissure;
Undermining stain of dentine shining up through
enamel.
Nine investigation sites appeared sound, seven had a
white spot lesion evident at the entrance to the fissure,
five had a brown spot at the entrance to the fissure and
nine sites had undermining stain of the dentine shining
up through the enamel. Thus these sites were chosen to
represent all clinically significant lesions and sound
sites.
Microfocal radiography
The teeth were sectioned facie-lingually to include the
investigation site. This was done using a low speed
diamond saw, under water (Labcut, Agar Scientific,
Stanstead, UK). The thickness of each section was
measured using a Digimatic micrometer (Mitutoyo,
Japan). The mean thickness of the sections was
0.67 mm (minimum 0.52 mm, maximum 0.94 mm). To
reduce the number of radiographs taken, a maximum of
six sections at a time were mounted alongside a high
precision 6 mm ball bearing to measure magnification
of the subsequent radiographic images to be taken.
A high definition microfocal X-ray unit described
previously by Buckland-Wright18 was used, which com-
prised a lanthanum hexaboride cathode and a single
electromagnetic lens which focuses the electron beam
onto an oil cooled multifaced tungsten target. The
X-ray source size ranges from 6 to 20,um, compared to
600pm for conventional dental X-ray units. The
mounted sections were placed close to the X-ray source
and the radiographic film was placed at some distance
away. This spatial arrangement can be altered depend-
ing upon the degree of magnification required and the
final distances were chosen in this study by making a
number of trial radiographs. The sections were placed
25 cm (approximately) from the X-ray source and the
film 2 m (approximately) from the sections. The micro-
focal X-ray unit was operated at 60 kVp, 0.95 mA and
exposed for 15 s and the radiographic films used were
Kodak Min-R Mammography film (Kodak, Hemel
Hempstead, Herts, UK) with a Min-R rare earth inten-
sifying screen. The latent images were immediately
automatically developed.
From the enlarged radiographic images or macro-
radiographs obtained, a subjective assessment of each
investigation site was made and categorised as:
Sound;
Caries confined to the enamel;
Caries extending into dentine.
 Ricketts et a/.: Histological validation for occlusal caries 91

Grev level value

0 I
0 50 100 150 200 250
Distance from surface / mm(xlO0)

Fig. 7. A captured image of a macroradiograph of a carious
extending into dentine, with investigation through the lesion.
In order to analyse the macroradiographs, they were
digitised using a high resolution charge coupled device
(CCD) camera (Kodak Videk Megaplus) which pro-
vides 1280 x 1024 square pixels with 256 grey levels and
a highly linear output. Using a Nikon Nikkor 55 mm
lens, films were digitised whilst backlit on a lightbox,
the light output of which was adjusted until the opti-
mum aperture (f/5.6 tofl8) of the lens could be used.
The digital output of the camera was connected to a
Univision UPXlOOO interface board and Univision
UDC2600 display controller, both in an IBM-
compatible 80486 PC, under the control of Optimas
Software (Bioscan Inc., USA). The image analysis
system was used to capture and investigate the resultant
images. Calibration of the image analysis system and
the degree of magnification was determined by measur-
ing the diameter of the 6 mm ball bearing. The enlarged
radiographic images or macroradiographs produced
were x 4 magnification. On the captured image a line
was drawn at right angles to the enameldentine junc-
tion (Fig. I), through what appeared to be the most
demineralised part of any lesion present. The width of
the line was set at five pixels so that grey level values
could be integrated across the line to overcome noise
and graininess of the film. Each point along the line was
assigned a grey level value in the range O-255 (O=black,
255=white) and a plot of these values against distance
from the surface of the tooth was obtained.
Such a plot can be seen in Fig. 2, where high grey
level figures represent radiopaque sites (light areas) and
low figures, radiolucent sites (dark areas). Thus point A
represents the grey level at the enamel surface of the
tooth, B to C represents the fall in grey level at the
enamel-dentine junction, C to D represents the gradual
increase in grey level at the advancing front of the
dentine lesion until sound dentine is reached at D.
The depth of lesion penetration was measured from
the grey level value by recording the distance of the
advancing front of the lesion, whether in enamel or
dentine, from the enamel-dentine junction (EDJ). The
advancing front of the lesion in enamel was taken as
Fig. 2. The resultant plot of grey level value against distance from
lesion the surface for the lesion seen in Fig. 7. A represents the grey level
at the surface of the tooth, B to C represents the fall in grey level at
the enamel-dentine junction, C the grey level in the carious dentine,
and C to D represents the gradual increase in grey level at the
advancing front of the dentine lesion until sound dentine is reached
at D.
being that point between the lesion and the EDJ at
which the grey levels were highest. This level was chosen
because in pits and fissures that have caries, the enamel
rarely reaches the degree of mineralisation as found in
sound buccal or lingual enamel. This may be because
the carious process affects the whole thickness of
enamel or that the carious process occurs at susceptible
sites, that have failed to undergo pre- and/or post-
eruptive maturation. In dentine, the advancing front of
the lesion was that point at which the grey levels
returned to those of the sound buccal and lingual tissue.
Confocal microscopy
The same examiner performed the analysis of the
macroradiographs and confocal microscopy, therefore
to eliminate examiner bias at least a month was allowed
to elapse between the two analyses. For confocal
microscopy each section was removed from its aqueous
storage medium and dried with a tissue. A saturated
solution of rhodamine B in tap water was then applied
to each cut surface in turn for 25 s, after which excess
was removed with a tissue. Reflection and fluorescence
images were then viewed with the confocal microscope
(Tandem Scanning Microscope (TSM), NORAN
INST, Middleton, WI, USA). An oil immersion objec-
tive ( x 10, 0.45 numerical aperture) was used without a
cover slip. For fluorescence imaging, the rhodamine dye
was activated by green light through an orange/red filter
to ensure only fluorescence from the dye contributed
to the images2. Images were either captured with a
Silicone Intensified Target (SIT) camera or recorded
directly onto 35 mm film.
From the fluorescence images the investigation sites
were categorised as:
Sound;
Caries confined to the enamel;
Caries extending into dentine.
 92 J. Dent. 1998; 26: No. 2
Fig. 3. (A) and (6) Fluorescence images of both sides
height 1.2 mm.
Digitised images were created of those sites with
caries using the MISIS 3DP image processing system
(MISIS Image, 1Old Rue de la Productique, 4,200
St Etienne, France). Measurements on the captured
confocal images were made from the deepest aspect of
any lesion toward the EDJ. The same microscopy
procedure was repeated on the reverse side of the
section and the mean value calculated.
STATISTICAL ANALYSIS
The Spearman Rank correlation coefficient was used to
determine the relationship between the two validation
techniques for estimating the distance of lesion penetra-
tion from the EDJ. Spearman correlation coefficients
from 0 to 0.3 were regarded as reflecting a weak
relationship, those from 0.3 to 0.7 a moderate relation-
ship and those from 0.7 to 1, a strong relationship.
Because correlation coefficients only assess the associ-
ation between two sets of measurements, the level of
agreement between the quantitative measurements
made with the two validation technique was also inves-
tigated by calculating the limits of agreement. Limits of
agreement were established according to Bland and
Altman21 by calculating the mean value of the two
of a single section, showing an enamel lesion. x10/0.45 oil immersion objective, field
different depth measurements for each lesion, and the
difference between the measurements. For visual assess-
ment of the likely variation between measurement tech-
niques, the differences were plotted against the means.
To calculate the differences, the depth measured from
the confocal image was subtracted from the depth
measured from the macroradiographs. Thus for dentine
caries, a positive difference indicated that the macro-
radiograph recorded a deeper lesion depth. The mean of
the differences (d) was calculated together with the
standard deviation (S.D.). The limits of agreement,
between which 95% of paired readings will vary, were
then represented by d- 1.96S.D. and d+1.96S.D. Limits
of agreement were calculated for measurements made
of dentine lesions and enamel lesions separately.
RESULTS
From the subjective interpretation of the macroradio-
graphs, six sites were sound, 10 had enamel caries and
14 had dentine caries. These results were in complete
agreement with those determined with the confocal
microscopy technique.
In the confocal microscope, the fluorescence images
of enamel and dentine caries appeared clear (Fig. 3 A
 Ricketts et al,: Histological validation for occlusal caries 93

Fig. 4. (A) and (6) Reflection images corresponding to the both sides of the section seen in Fig. 3A and 6. x1010.45 oil immersion objective,
field height 1.2 mm.

and B), however, contrast between normal and diseased

tissue in the reflection images were rather unsatisfactory
because of the structural changes due to caries (Fig. 4 A
and B). In the reflection images lesion boundaries in
enamel could be determined by eye but were difficult to
quantify. In dentine the lesion was not obvious because
of breakdown in tubular structure which contributes to
the normal reflection image. Absence of dentine struc-
ture could be used to indicate carious tissue but this
would again be difficult to quantify with image process-
ing techniques. Thus, fluorescent images only were used
for quantitative analysis, because the dye was well
absorbed both by the demineralised enamel and espe-
cially the demineralised dentine. The use of the appro-
priate pass filter gave good contrast for easy image
capture and manipulation.
Two sections with extensive dentine caries were
damaged on sectioning, which only allowed depth
Fig, 5. Macroradiograph showing the image of the enamel lesion
measurements into dentine to be calculated. The seen in Figs 3 and 4.
macroradiographs provided a single measurement for
caries depth within a single section (Fig. 5). However,
using the confocal technique, differences in lesion depth from the macroradiograph (Fig. 6). Despite this there
were noted depending on which side of the section was was a strong positive relationship between depth
examined in the microscope (Fig, 3 A and B). A mean measurements determined from both histological tech-
value was therefore calculated which, for dentine niques (~0.93, P<O.OOl). When the distances of the
lesions, was consistently less than the equivalent result enamel lesions from the EDJ were compared for
94 J. Dent. 1998; 26: No. 2

1
1 E 0.8
l Dentine lesions
. .
Q Enamel lesions . ,o 0.6 .
. . .
.
Mean difference for dentine lesions
5 0.4
21 .4 .
. . Yi
ST l
4 5E 0.2 .
. 4

. g! 4 l Mean difference for enamel lesions

4 2 0 u
4 v
$
5E o4
-0.2

i o::,::,. ,.,,
B 8
.G
a
-0.4
l Dentine lesions
-1
s8 -0.6
0 0.5 1 1.5 n
L Q Enamel lesions
Distance of lesion from EDJ (MR) I mm ziii -0.8
n
Fig. 6. The relationship between the distance of the advancing front
of the lesion from the enamel-dentine junction (EDJ) as measured -1 I

from the macroradiograph (MR) and the confocal image (CF). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Mean of depth measurements made by MR and CF / mm
Fig. 7. The difference in depth measurements made from macro-
both histological techniques only a moderate positive radiographs (MR) and the confocal fluorescent images (CF), plotted
relationship was found (r=0.65, P=O.O42) (Fig. 6). against the mean of the paired measurements. The solid upper
most horizontal line is the mean of the differences in depth
A plot of the differences between depth measurements
measurements of dentine caries and the lower broken line, of
made from the macroradiographs and the confocal enamel caries.
images, and the mean of the paired readings can be seen
in Fig. 7. For dentine lesions the mean difference was
dimension. This is supported by the fact that the
0.41 mm (S.D.=O. 165) and the limits of agreement were confocal images varied, depending from which side of
+0.73 and +0.09. For enamel lesions, the mean of the
the section the lesion was observed. However, it is in the
differences was 0.075 mm and the limits of agreement authors opinion that the investigation line drawn on
were +0.408 and -0.258. the macroradiograph represents a suitable compromise
and the depth measurements made using this technique
correlated well with those taken with the confocal
DISCUSSION
technique.
The high definition microfocal technique used in this Computer aided image analysis of the macroradio-
study has been previously described for the clinical graphs also has the potential for quantitatively assess-
study of arthritides, metabolic disorders and some other ing the degree of mineral loss within the carious lesion,
bone diseases2. Its use as a histological validating by expressing the grey level values within the enamel
technique for dental caries has recently been described and dentine lesions as a percentage of that for sound
by Ricketts et a1.19. It allows the object under investi- tissue from the same section. It must be appreciated
gation to be placed close to the point X-ray source and that this does not measure the absolute mineral loss, as
the film at a distance, and because the X-ray source is so in a chemical analysis, but does serve as a good guide.
small (approximately 6-20,~~rn) an image is produced Such quantitative assessment cannot be made using
without the blurring associated with the penumbra confocal microscopy, by assessing the intensity of
effect. The degree of magnification can simply be fluorescence and as such this issue was not pursued in
adjusted by altering the X-ray source, object and film this study.
spatial arrangement. With the use of mammography Both validation techniques agreed on the presence
film the spatial resolution has been reported to be and extent of caries penetration made from a subjective
excellent, allowing objects as small as 70 pm diameter to assessment of the images. Unlike the microfocal X-ray
be detected with the use of x 10 magnification*. unit, confocal microscopes are more widely available in
In this study the macroradiographs were subjectively research institutes. Confocal microscopy has been used
assessed and quantitatively analysed to measure lesion with fluorescent dyes for the histological validation of
depth. The investigation line was drawn through the approximal caries23 but to date has not been used for
most demineralised area of enamel and as such, depth occlusal caries.
measurements made from this, assumes a certain degree The Tandem Scanning type of microscope has been
of symmetry of the lesion. This is of course not always described in detail by Watson and Boyde20,24. Essen-
true, and it must be remembered that the macroradio- tially this technique enables thin optical sections, or
graph is a two-dimensional image of a thick section of tomograms, to be made below the surface of a semi-
tooth and a degree of asymmetry will exist in the third transparent material with improved resolution. The

depth to which the microscope is able to scan beneath
the cut surface of the section depends upon the trans-
parency of the material. For enamel, this depth is about
100 pm for most oil immersion objectives with no cover
slip (to increase the working distance of the lens). Using
a high numerical aperture lens the thickness of the
tomogram is less than 1 pm. Thus this microscope has
the potential to look beneath the surface of a thick
section without destroying it. Problems of specimen
preparation associated with the thin sections required
for conventional transmitted light microscopy
therefore avoided.
The sections cut in this study were approximately
670pm thick. Complete sampling of the whole section
was therefore not possible using the confocal technique,
however, serial grinding and imaging techniques could
have been used to build up a composite image of an
entire lesion, but this would have greatly increased the
time required. The mean difference in depth measure-
ments made from the macroradiographs and the con-
focal fluorescent images was always positive for dentine
lesions (Fig. 7). This means that depth measurement of
the fluorescent images of dentine caries obtained in this
study were generally less than the depth measured from
the macroradiograph, the average difference being
0.41 mm. Thus either the analysis of the macroradio-
graphs over-estimated the depth of the lesion or the
confocal fluorescence image under-estimated its extent.
Increasing the soaking time in the dye could have
over-come this discrepancy as dye penetration
inevitably be a function of time. The differences
between measurements of enamel lesions was not so
great.
Classically, histology has been used to validate caries
diagnosis, because it is more accurate than visual ex-
amination of the intact tooth for example. However, the
more accurate a diagnostic technique becomes the more
accurate must be the histological technique. It is poss-
ible that with the newest of diagnostic techniques, such
as electrical conductance methods of diagnosis, that
inaccuracies of the diagnostic technique may only re-
flect the inability of the validation technique to detect
such small lesions25. This dilemma is true when com-
paring validation techniques, there lacks an absolute
true state or gold standard. Failure of one technique
to identify caries may only be a result of over-
prescription by the other. Thus this study only com-
pares validation techniques, but can not address
absolute accuracy. This study supports that of a pre-
vious study which has shown that the method of
validation could affect the quantitative assessment of
caries, however, the impact of validator cannot be
assessed as only one examiner looked at the samples in
this study.
Both the histological validating techniques used in
this study enabled a comparatively quick assessment of
the presence or absence of caries, as well as the quanti-
tative assessment of lesion depth. However, both tech-
Ricketts et al,: Histological validation for occlusal caries 95
niques identify demineralisation only. It is known that
demineralisation precedes bacterial infection and it is
this infection of the tissue which should prompt opera-
tive intervention. It is therefore important that future
research into validation techniques addresses the
infectivity of the tissue, in freshly extracted teeth.
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