JOURNAL CLUB: NON INVASIVE IN-VIVO CONFOCAL LASER SCANNING MICROSCOPY FOR EXAMINATION OF GINGIVAL TISSUES

Non-invasive in vivo imaging by
confocal laser scanning
microscopy of gingival tissues
following natural plaque
deposition
Jörg Eberhard, Hendrik Loewen,
Alexander Krüger et al.
J Clin Periodontol 2014; 41: 321–326
Shilpa Shivanand
II MDS

Introduction
• Constantly forming bacterial deposits on the teeth 
Chronic inflammatory response 
Subclinical gingivitis 
Irreversible and destructive periodontitis
(Pihlstrom et al 2005)

Confocal Laser Scanning Microscopy
(CLSM)
• The basic concept of confocal microscopy was originally
developed by Marvin Minsky in the mid 1950s.
• Used for years to study biofilms and biomaterials in vitro in
various biological systems
(Neu et al 2010)
• Recently introduced for the evaluation of the corneal
epithelium in patients
(Zhivov et al 2009 Reichard et al 2010)

• Confocal microscopy offers several advantages over conventional
wide field optical microscopy, including
- the ability to control depth of field
- elimination or reduction of background information
away from the focal plane (that leads to image degradation)
- the capability to collect serial optical sections from
thick specimens
• The basic key to the confocal approach is the use of spatial filtering
techniques to eliminate out-of-focus light or glare in specimens
whose thickness exceeds the immediate plane of focus.

 Comparison of wide
field (upper row) and
laser scanning
confocal fluorescence
microscopy images
(lower row).
 (a) and (b) Mouse
brain hippocampus
 (c) and (d) Thick
section of rat smooth
muscle
 (e) and (f) Sunflower
pollen grain tetrad
auto fluorescence.

• In dental research used to characterize structure of carious
lesions in hard substances and biofilm formation on tooth
surfaces after staining ex vivo
(Auschill et al 2005, de Carvalho et al 2008)
• Until now no non-invasive imaging methods are available that
are capable to show early pathologies of the gingiva or mucosa
adjacent to teeth or implants in humans
(Xiang et al 2010)

• Established imaging methods like radiographs  not capable
to document histological changes of soft tissues, because
low resolution and the low density of targeted tissues.
• Imaging with CLSM in contrast generates high-resolution
images of tissues and may be well suited for clinical research
or diagnostic purposes.

Principle Of CLSM
• A confocal microscope creates
sharp images of a specimen
that would otherwise appear
blurred when viewed with a
conventional microscope.
• This is achieved by excluding
most of the light from the
specimen that is not from the
microscope’s focal plane.
• The confocal microscope
incorporates the ideas of
point-by-point illumination of
the specimen and rejection of
out-of-focus light.

Subjects
• Ten subjects (female = 6, male = 4) were examined for oral
health and pre-existing periodontal diseases.
• Excluding criteria were: (i)periodontal disease (ii)smoking
(iii)systemic diseases.
• Subjects eligible were informed about the aim and protocol of
the study and signed an informative consent.

Study Protocol
• All participants received professional dental prophylaxis to
remove plaque deposits and to reduce any gingival
inflammation 7 days before baseline.
• At baseline, the participants were advised to terminate tooth
brushing of the upper canine and incisors for a period of 7
days.
• Antibacterial mouth rinses were not allowed.

• At baseline and at day 7, dental plaque was recorded by the
Oral Hygiene Index (OHI).
• One examiner recorded at six sides per tooth bleeding on
probing (BOP) using a periodontal probe.
• All clinical measurements were done after CLSM procedures.
• After 7 days, all teeth were professionally cleaned and oral
hygiene was resumed.

CLSM Imaging
• The in-vivo CLSM used was based on the Heidelberg Retina
Tomograph (HRT-II, Heidelberg Engineering GmbH,
Heidelberg, Germany) in combination with a lens system
attachment known as the Rostock Cornea Module (RCM).
• The system equipped with a red laser light source of 670 nm
provides a motorized z-movement for selection of the confocal
image plane.

• The contrast is given by backscattering light from different
tissue constituents (cells, membranes, organelles) which was
collected in reflective mode by a photomultiplier tube behind a
confocal pinhole.
• Z-stacks of two-dimensional en-face images were acquired
with a size of 384×384 pixels (pixel size of 0.8×0.8µm) and an
inter-slice distance of 0.9µm.

• The RCM used a water immersion objective with a high
numerical aperture and a long working distance (Achroplan,
magnification 63x, numerical aperture 0.95, water immersion
working distance 1.45mm, Carl Zeiss GmbH, G€ottingen,
Germany).
• For connecting the objective with the tissue, a PMMA cap was
used.
• Coupling between the tissue and the contact cap was facilitated
with a thin lubricant layer of Vidisic gel.

Imaging
• Subjects were placed in a chinrest in front of the objective.
• The objective was moved to direct the focus on the upper
papilla between the primary incisors of the participants.
• After the objective was gently placed in maximal proximity to
the papilla, the sulcus was systematically examined from the
most coronal aspect of the papilla to the most apical part of the
gingival sulcus that was accessible with the confocal
microscope tubes.

• During screening, images were acquired by the CLSM and
stored continuously with 30 fps.
• Thus, approximately 800– 1200 images were created during
each examination.
• During post-processing, a number of representative images
were selected and analyzed.

In vivo CLSM imaging at baseline and
day 7
• Discrimination between tooth hard substances, cells and
plaque deposits were possible.
• Cell membranes of epithelial cells were apparent and
cytoplasm's as well as cell nucleus were visible.
• Plaque deposits were visualized as structured signals adhering
to the tooth hard substances.
• Single bacteria could not be identified.

• At baseline, the sulcular epithelial cells appeared closely
adherent to each other showing a regular surface towards the
gingival sulcus and a few floating epithelial cells were
observed.
• From the most coronal aspect of the gingival margin, the
epithelial cell layer penetrates 500–800µm apically and
became closely adsorbed to the tooth surface, indicating the
coronal border of the junctional epithelium.

• At day 7 of plaque accumulation, the epithelial cells showed a
more irregular appearance compared to the baseline images.
• Within the epithelial cell layer black areas were frequently
observed and also the epithelial cell volume increased in some
views.
• The degree of epithelial cell displacement showed inter-
individual differences, however, increased numbers of epithelial
cells were observed within the gingival sulcus

• In addition, the distance between the most coronal and apical
aspect of the epithelial cell layer became widened, indicating
an apical penetration of the sulcus epithelium.
• The gingival sulcus was partly filled with round cells of
approximately 10µm in diameter, single floating epithelial
cells and plaque deposits.

• Massive plaque deposits approximately 50µm thick were
adherent to the tooth surfaces in delimited areas or appeared as
thin layers of only a few micrometers.
• The plaque deposits were located within the gingival sulcus
coronal the junctional epithelium showing an irregular surface.
• Single bacteria were not discriminated.

Discussion
• The present study demonstrated for the first time the application of
CLSM for the in vivo imaging of histological changes of the gingival
sulcus and adjacent tissues induced by naturally formed bacterial
plaque deposits.
• In comparison from baseline to day 7 of declined oral hygiene at
selected teeth, the images showed:
(i) increased epithelial cell irregularities
(ii) apical penetration of the sulcus epithelium
(iii) increased frequency of cellular infiltrates within the sulcus
(iv) plaque deposits adherent to dental hard tissues.

• The clinical parameters clearly indicated that the increasing
plaque deposits on the tooth surfaces induced an inflammatory
reaction of the gingival tissues.
Loe et al 1965
• The observed inflammatory reaction displayed the initial phase
of gingivitis, which is characterized by the initial increased
flow of gingival crevicular fluid and high frequency of
inflammatory cells within the gingival sulcus  condition
also designated as the “normal, healthy gingiva”
Kinane & Lindhe 2003

• The apical penetration of the sulcular epithelium and the
following increase in distance between the gingival margin
and the coronal part of the junctional epithelium was shown in
the present CLSM images.
• Bacterial plaque deposits observed at day 7 and the epithelial
cell layer of the sulcus were always separated by a narrow
liquid filled space.
• After 7 days of plaque formation the gingival sulcus was filled
with round shaped cells and due to their diameter of
approximately 10µm it was likely that these cells were
leucocytes.

• Leucocytes penetrate the junctional epithelium and migrate
into the gingival sulcus to exert defense mechanisms against
bacteria
Thurre et al 1984, Ebersole 2003
• Images at day 7 showed black voids having the size of
epithelial cells. Because black parts of the images indicated the
presence of water, it is likely that these areas represent
inflammatory edemas', which have been described also in
earlier reports
Schroeder et al 1973

• In dentistry, confocal microscopy was used for the analysis of
dental biofilms and biomaterials in vitro and enables high-
resolution images to be made of samples with minimum
requirements for preparation.
• Currently CLSM is a favorable research tool in situations,
which deserves a contact-free imaging tool for the longitudinal
observation of histological processes in periodontology and
peri-implant dentistry.

Conclusion
• The present study showed that CLSM is suitable for in vivo
imaging of the microscopic appearance of the gingival sulcus
and nearby tissues.
• In vivo imaging by CLSM may be a contact-free research tool
for longitudinal studies of pathological, healing or regenerative
processes and is today limited due to the dimensions to the
anterior region of the oral cavity.

I . Microscopic features of enamel and dentinal caries under
confocal laser scanning microscopy (CLSM) and image
analyzer: preliminary experimental study.
The Medical journal of Malaysia 62:3 2007 Aug
Abstract:
• This study was designed to identify surface and subsurface
microscopic changes in different carious lesions by using
Confocal Laser Scanning Microscope (CLSM) and Image
analyzer (light microscopy). Thirty extracted carious posterior
teeth were fixed, embedded and polymerized in plastic fixation
medium.
• The final thin sections (80mm) were stained with H&E and
Masson Goldner's Tricome while others were left unstained.

• Under Confocal, marked differences between control sound
enamel and dentin, and carious area of the samples were
observed which illustrated that a correlation existed between
the zone of autofluoresence, demineralization and carious
enamel and dentin.
• Compared to CLSM, Image Analyzer only produce two
dimensional images but the histopathological changes were
better appreciated by using various staining methods.

II. Confocal Microscopy for Real-Time Detection of Oral Cavity
Neoplasia
Clinical Cancer Research October 15, 2003
• Purpose: The goal of this study was to characterize features of
normal and neoplastic oral mucosa using reflectance confocal
microscopy.
• Experimental Design: Oral cavity biopsies were acquired from
17 patients who were undergoing surgery for squamous cell
carcinoma within the oral cavity. Reflectance confocal images
were obtained at multiple image plane depths from biopsies
within 6 h of excision. After imaging, biopsies were fixed in
10% formalin and submitted for routine histological examination

• Reflectance confocal images were compared with histological
images from the same sample to determine which tissue
features contribute to image contrast and can be potentially
imaged using in vivo confocal microscopy.
• Conclusions: Results support the potential for this tool to play
a significant role in the clinical evaluation of oral lesions, real-
time identification of tumor margins, and monitoring of
response to therapeutic treatment.

JOURNAL CLUB: NON INVASIVE IN-VIVO CONFOCAL LASER SCANNING MICROSCOPY FOR EXAMINATION OF GINGIVAL TISSUES

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JOURNAL CLUB: NON INVASIVE IN-VIVO CONFOCAL LASER SCANNING MICROSCOPY FOR EXAMINATION OF GINGIVAL TISSUES

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