SHORT COMMUNICATION

In vivo imaging of enamel by reflectance confocal microscopy(RCM): non-invasive analysis of dental surface

Maria Contaldo • Rosario Serpico • Alberta Lucchese

Received: 10 December 2012 / Accepted: 16 March 2013

� The Society of The Nippon Dental University 2013

Abstract The aim is to establish the feasibility to image

in vivo microscopic dental surface by non-invasive, real-

time, en face Reflectance Confocal Microscopy (RCM).

Fifteen healthy volunteers referred at the Multidisciplinary

Department of Medical-Surgical and Odontostomatological

Specialties, Second University of Naples, Naples, Italy,

were enrolled. A commercially available hand-held RCM

(Vivascope�3000, Lucid, Rochester, NY, USA) was used to

image in vivo the dental surface of the upper right and left

central incisors of each volunteer. Totally, thirty vestibular

surfaces of upper central incisors were imaged in vivo by

RCM to preliminary image the dental surface and assess the

feasibility of a more extended study on teeth. In vivo RCM

was able to image the dental surface within the enamel, at a

maximum depth imaging of 300 lm, with images good in

quality and the capability to detect enamel structures such as

enamel lamellae and enamel damages, such as unevenness

and cracks. In conclusion, enamel ‘‘optical biopsy’’, gained

by RCM imaging, revealed to be a non-invasive real-time

tool valid to obtain architectural details of the dental surface

with no need for extraction or processing the samples. RCM

appears to be an optimum auxiliary device for investigating

the architectural pattern of superficial enamel, therefore

inviting further experiments aimed to define our knowledge

about damages after etching treatments or bracket removal

and the responsiveness to fluoride seals and the morphology

of the tooth/restoration interface. Moreover, this device

could also be used to detect relevant diseases like caries, or

to assess surface properties to evaluate lesion activity.

Keywords In vivo dental imaging � Enamel structure �Reflectance confocal microscopy � In vivo RCM � Dental

surface

Introduction

To date, microscopic dental examination requires ex vivo

elaborate techniques. Extracted teeth need to be decalci-

fied, processed and cut in thin slices to be available for

microscopic analysis [1]. These procedures are time and

money consuming, considering time and costs spent to

complete the procession of the sample. Therefore, alter-

native techniques could expand these limitations and avoid

the need for extraction and histopathological processes.

The literature reports several alternative imaging approa-

ches to dental analysis, such as scanning electron micros-

copy (SEM) [2] and optical coherence tomography (OCT)

[3]. However, these techniques still require to extract and

assess the tooth for its analysis.

Confocal microscopy (CM) is a microscopic imaging

technique that allows to avoid the sample preparation due

to its ability to optically scan on horizontal planes, layer by

layer, both specimen and living tissues along their depth

[4]. The result is a 2-dimensional image of a horizontal

virtual section of the tissue, which displays architecture and

cellular details of the section at microscopic resolution.

CM has been applied by several authors to study oral-

maxillary structures both in vivo and ex vivo [5]. Authors

such as Watson et al. [6] and Burmeister et al. [7] pre-

liminarily investigated the in vivo confocal microscopy

application on dental surface imaging, while such authors

as Ogaard et al. [8] and Lucchese et al. [1] reported their

experiences in describing teeth structures and compounds

by ex vivo confocal microscopy.

M. Contaldo (&) � R. Serpico � A. Lucchese

Multidisciplinary Department of Medical-Surgical and

Odontostomatological Specialties, Second University of Naples,

Via Luigi De Crecchio 6, 80138 Naples, Italy

e-mail: maria.contaldo@gmail.com

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Odontology

DOI 10.1007/s10266-013-0110-9

In detail, the so-called ‘‘in vivo reflectance confocal

microscopy’’ (RCM) is based on the mechanism of back-

scattering of light. The refracted light is emitted by a

substance in the living tissue after an incident light at a

specific wavelength hits it. At present, in vivo RCM is

widely used in dermatology for in vivo microscopic

imaging of the skin in the clinical practice as diagnostic

and follow-up adjunct [9], and recent works have shown its

usefulness in oral mucosa imaging too [10].

Aim of the present study is to evaluate the feasibility to

image microscopic architecture of dental surface using an

in vivo RCM commercially available for skin imaging,

which revealed its usefulness in imaging oral mucosae in a

non-invasive, painless, real-time way [10].

Subjects, materials and methods

Subjects

Fifteen healthy volunteers (8 males, 7 females, mean age

38.4 ± 16.2 years) enrolled at the Multidisciplinary

Department of Medical-Surgical and Odontostomatological

Specialties, Second University of Naples, Naples, Italy,

after signed consent, were subjected to in vivo RCM

examination of their upper central incisors vestibular sur-

faces (permanent teeth) to establish the usefulness of

in vivo RCM to image the dental structures. Totally, 30

upper central incisors were imaged, and classified at clin-

ically inspection as healthy teeth (26) and teeth with

cracked enamel surfaces (4). No teeth were successively

extracted.

In vivo reflectance confocal microscope

A commercially available hand-held RCM (Viva-

scope3000�, first version, Lucid, Rochester, NY, USA)

was applied [10]. The system operates with a diode laser

class 3A (European version) at 830 nm wavelength and

with a 309 water immersion objective lens with a

numerical aperture of 0.9. The laser power can vary in the

range of 5–10 mW according to the layer to evaluate and it

causes neither tissue damage nor dental heating. In order to

obtain confocal images of the tissue from the plans parallel

to the RCM lens (confocality), the device needs to work in

close contact to the dental surface. Ultrasound gel is

interposed between the lens and the window, and the

window and the mucosa, to optimize the incident and the

refracted lights transmission. Images are displayed in

500 9 500 lm horizontal sections, 5 lm in thickness and

with 0.5–1 lm lateral resolution. These parameters are

similar to standard histology; thus, offering the advantage

to visualize the cellular details [11].

RCM acquisition method

En face, single, 500 lm 9 500 lm RCM vertical stacked

images were collected starting from the top and progress-

ing deeper until reaching the last visible dental structures.

The microscopic parameters identified by RCM were cor-

related with the equivalent ones reported ex vivo in liter-

ature. These parameters are similar to standard histology;

thus, offering the advantage to visualize the microscopic

details. Because of its rigid probe, currently the device is

not allowed to reach the posterior and lingual regions of the

teeth. For these reasons, access is limited to the vestibular

surface of central incisors.

Results

Healthy dental surface

The optical horizontal sections of the dental surface were

easily observed by RCM. The RCM images of healthy

enamel showed a not perfectly smooth surface: it was

possible to observe furrows and unevenness of variable

depth and width with smoothed edges along with areas of

smoother enamel (Fig. 1a).

Starting from 10 lm underneath the surface and going

deeper toward the dentoenamel junction, the peculiar

structure of the enamel appeared: densely packed enamel

rods were embedded in interprismatic enamel. In detail,

strongly bright roundish areas, regular in size and geo-

metrically disposed, corresponded to the interprismatic

substance, highly refractive; these refractive areas con-

toured the enamel prisms, deductively recognized as very

dark and well-defined geometric circles (diameter less than

1 lm) regularly disposed and limited by the interprismatic

substance (Fig. 1b). Going deeper, the regular architecture

was preserved and the images remained good in quality and

contrast. 280 lm underneath the surface, some strongly

dark linear figures were identifiable, parallel to the hori-

zontal plane and interrupting the geometric enamel struc-

ture. They were related to the enamel lamellae, the

microscopic separations in the enamel which extend

inward from the enamel surface for varying distances and

which are filled with organic material (Fig. 1c). The den-

tin-enamel junction was not imaged. The maximum depth

reached was about 300 lm.

Cracked enamel surface

When the cracked enamel surface was imaged, among the

regular prismatic and interprismatic structures previously

described for healthy teeth, a quite pronounced fracture

was clearly identifiable because of the presence of a dark

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cleft irregular in shape, limited by pale bright walls,

brighter in some points (Fig. 1d). In 3 out 4 cases, the

fracture was visible until the last frame captured, 300 lm

underneath the surface.

Discussion

The microscopic study of dental structure and defects is

actually based on ex vivo techniques, which require the

tooth needs to be extracted and processed by long and

intricate procedures.

In vivo confocal microscopy is a non-invasive imaging

technique, which allows to obtain microscopic details of

the living tissues in real time, with no need for biopsy or

anesthetics [12]. In vivo RCM, based on the mechanism of

backscattering of light, is largely used in dermatology as

adjunct tool for the clinical management and therapeutic

monitoring of patients affected by melanocytic and non-

melanocytic skin tumors and inflammatory skin diseases

and it represents both an alternative to conventional his-

topathological examination and a tool able to orient clini-

cian and surgeon towards the most representative sites to

biopsy [13]. In vivo RCM has been also applied in

Fig. 1 a Dental surface, not perfectly smooth but with furrows and

unevenness of variable depth and width. b Enamel structure, regular

interprismatic substance (bright polygonal areas) and enamel prisms

(dark areas among the interprismatic substance). c Enamel lamellae,

280 lm underneath the surface, appeared as strongly dark linearfigures parallel to the horizontal plane and interrupting the geometric

enamel structure. d Cracked enamel was identified as a dark irregular

cleft, limited by bright boundaries. Scale bar 100 lm

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stomatology to image the healthy oral mucosa, thus pro-

viding a comprehensive description of the confocal criteria

visible in healthy mucosa [10].

Thanks to the preliminary works exposed by Watson [6],

for the first time it was possible to image in vivo at micro-

scopical resolution, on en face optical sections, the teeth and

the oral mucosa, with neither anesthetic injection nor pain

for the volunteer. The deep enamel structure was easily

observed and distinguished from the resin composite resto-

rations and the presence of voids which were not at all

clinically obvious on the surface of the restoration was also

imaged. The lamellae in the enamel were visible as well as

enamel prisms and possibly dental plaque. Watson intuited

the versatility and the benefits of using this kind of device to

study clinical conditions. The importance of this pioneer

work is indisputable; however, in Watson’s work the exact

depth of view could not be recorded and the pictures were not

well-defined in contrast and in details resolutions. Thanks to

the engineering and software developments occurred during

the last two decades, RCM devices commercially available

now allow to standardize the operative protocol and to

improve quality of contrast and magnification.

In the present work, we performed in vivo teeth surface

microscopic imaging in a non-invasive real-time approach

using RCM to test its clinical potential in superficial teeth

imaging.

The examinations were performed without trauma or

damage for the subjects enrolled. The subjects well toler-

ated the short time spent to perform the imaging (mean

time 5 min) and the non-invasive, relatively simple

procedure.

In addition, RCM does not need stain injection, neither

rinse solution nor topic substance application, except the

gel used for allowing a good light transmission from and to

the microscope, since it is based on the intrinsic, refractile

proprieties of the subcellular structures and molecules.

Based on this property, RCM can be used without risk of

allergic/irritant reactions to stains and/or postoperative

complications, and as it is an ionizing radiation-free device,

it can be used in safety in children and pregnant.

Despite the ex vivo approach, the in vivo RCM imaging

allows to analyze the dental surface without processing the

tooth in a clinical condition close to the real contest of the

tooth, thus avoiding to image iatrogenic enamel scratches

such as the ones that may occur after extraction, and with

the real hydration state of the tooth in its natural

environment.

Our results demonstrated high correspondence between

in vivo RCM and ex vivo CM images collected by other

authors [8].

The device used in the present study has been able to

observe several enamel layers well in quality and contrast,

until 300 lm underneath the surface in real time with no

need to extract the teeth. Since the thickness of the enamel

is between 1.5 and 2.5 mm but the maximum depth

reached by RCM is 300 lm, the sole dental structure

imaged by RCM may be the enamel, while dentin-enamel

junction was not imaged.

To date, the access by the RCM commercially available

was unfortunately limited to the vestibular surface of the

upper central incisors because of the ergonomics of the

device, born for skin imaging in dermatology. Indeed,

further efforts must be made to optimize the device ergo-

nomics thus allowing the examination of all anatomical

dental surfaces, until the third molars. RCM teeth evalua-

tion also presented penetration limits through dental deeper

layers because of light backscattering due to superficial

enamel hyper-refractivity.

Despite these limitations, the preliminary results on

RCM dental surface imaging encourage carrying out a

more extended study in vivo on teeth. This technology

could offer support to clinical management of patients with

dental diseases or damages and could help to understand

in vivo the damages related to etching and bracketing. In

the clinical practice, a number of different situations

require the removal of a thin layer of enamel and some-

times it is cut-off by traumatic or iatrogenic causes. The

in vivo study of the effects of several substances on enamel

surfaces could help industries to produce less harmful

materials to teeth and gives the opportunity to identify and

exploit the effects of rotary tools on enamel, thus allowing

for a correct finishing technique.

The present work was a feasibility study developed to

establish the capability of a reflectance confocal micro-

scope commercially available for in vivo skin imaging to

in vivo image enamel tissue. The preliminary results invite

further experiments aimed to define our knowledge about

in vivo damages after etching treatments, or bracket

removal and the responsiveness to fluoride seals and the

morphology of the tooth/restoration interface, as well for

imaging the oral mucosae. Moreover, this device could also

be used to detect relevant diseases like caries, or to assess

surface properties to evaluate lesion activity [14, 15].

Nevertheless, studies on pathological conditions affect-

ing the teeth are justified and possible. Obviously, further

efforts must be done to improve the device, thus allowing

to image also other dental surfaces such as the occlusal and

palatal ones, and all types of teeth.

In conclusion, RCM appears to be a good auxiliary

device for investigating in vivo the microscopic architec-

ture and features of enamel by a non-invasive way and a

real-time response, thus dramatically reducing the timing

of the dental imaging and obtaining microscopic details of

the living tissues with no need for teeth extraction.

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Conflict of interest The authors declare that they have no conflict

of interest.

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