Non-invasive in vivo imaging by

confocal laser scanningmicroscopy of gingival

tissuesfollowing natural plaque

depositionJörg 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 andday 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.

CROSS REFERENCE

I . Microscopic features of enamel and dentinal caries underconfocal 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.