Confocal Laser Scanning Microscopy: A Nondestructive Subsurface Histotomography of Healthy Human Bone

Abstract

Microscopy of bony tissue usually requires special treatment for decalcification and processing of thin sections. Confocal laser scanning microscopy (CLSM) allows the nondestructive histotomography of organic hard tissue. The aim of this study was to visualize healthy human bone structures and to correlate identical areas in CLSM and conventional light microscopy. Each sample of healthy human lower jaw (n = 20) was divided into three parts: (1) fresh, untreated bony blocks studied by CLSM; (2) MMA-embedded thin sections (without decalcification), HE stained and studied by CLSM and conventional light microscopy (correlation of identical areas); (3) decalcificated, HE stained, histological sections studied by conventional light microscopy. In untreated bony blocks, microstructures such as osteocytes and lamellae were identified by CLSM. These structures could be correlated with conventional light microscopy. In CLSM, subcellular structures cannot yet be interpreted, whereas cytoplastic processes of osteocytes were seen with high contrast. With CLSM, nondestructive histology of cortical bone can be obtained. The risk of artifacts due to pretreatment is minimized, and subsurface visualization does not affect the interpretation.

Comparative histomorphometry and resonance frequency analysis of implants with moderately rough surfaces in a loaded animal model

“…After sacrificing the animals, the implant–bone blocks were fixed in 50% ethanol and prepared for histology as described earlier (Donath & Breuner 1982) including embedding in PMMA (Technovit ® 7500; Kulzer, Hamburg, Germany). Inspection of subsurface areas of the bone/implant interface (older: boundary layer) by confocal laser scanning microscopy (CLSM) allowed the use of unsectioned and not stained block samples (Grötz et al 1999; Al‐Nawas et al 2003). Thus, the implants were cut in the mesio‐distal direction, ground to a grain of 1200 and polished.…”

“…CLSM allows visualizing high‐contrast images of the BIC area without sectioning or staining. The method has been validated in various studies (Grötz et al 1999; Al‐Nawas et al 2003). The unstained blocks were then inspected by CLSM (Leica TCS SP2 X1: reflection mode, Leica, Wetzlar, Germany) under 633 nm laser light illumination and oil immersion objectives (× 10 magnification), resulting in an image size of 1.5 × 1.5 mm.…”

Correlating micromorphology of the pulpal odontoblast seam using confocal laser scanning microscopy and dark field microscopy

“…It has been shown that confocal laser scanning microscopy (CLSM) allows subsurface visualisation of nondecalcified biological hard tissue, thus reducing the problem of artefacts due to surface irregularities following the grinding process [8,9]. Being an innovative technique of histotomographic visualisation of vital osteocytes, CLSM findings have been validated by comparison of identical sample areas by standard dark field microscopy (DFM) and cell labelling with Bisbenzimide [10]. This study reports on the visualisation of the odontoblast seam by the CLSM technique and again with DFM and fluorochrome (Bisbenzimide) labelling as reference.…”

Methods of Evaluation for Bone Dimensions, Densities, Contents, Morphology, and Structures

“…Stepwise movement permits an artifact-free, topographic image to be obtained in a nondestructive approach without the use of special staining. 45 Stored multilayer 2D images can then be reorganized to show 3D or cross-sectional pictures. CLSM has been used for viewing the structures at the implant-tissue interface, such as unmineralized bone matrix or mineralized bone.…”