Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Virtobot--a multi-functional robotic system for 3D surface scanning and automatic post mortem biopsy

The Virtopsy project, a multi-disciplinary project that involves forensic science, diagnostic imaging, computer science, automation technology, telematics and biomechanics, aims to develop new techniques to improve the outcome of forensic investigations. This paper presents a new approach in the field of minimally invasive virtual autopsy for a versatile robotic system that is able to perform three-dimensional (3D) surface scans as well as post mortem image-guided soft tissue biopsies.

Accident or homicide - virtual crime scene reconstruction using 3D methods

The analysis and reconstruction of forensically relevant events, such as traffic accidents, criminal assaults and homicides are based on external and internal morphological findings of the injured or deceased person. For this approach high-tech methods are gaining increasing importance in forensic investigations. The non-contact optical 3D digitising system GOM ATOS is applied as a suitable tool for whole body surface and wound documentation and analysis in order to identify injury-causing instruments and to reconstruct the course of event. In addition to the surface documentation, cross-sectional imaging methods deliver medical internal findings of the body. These 3D data are fused into a whole body model of the deceased. Additional to the findings of the bodies, the injury inflicting instruments and incident scene is documented in 3D. The 3D data of the incident scene, generated by 3D laser scanning and photogrammetry, is also included into the reconstruction. Two cases illustrate the methods. In the fist case a man was shot in his bedroom and the main question was, if the offender shot the man intentionally or accidentally, as he declared. In the second case a woman was hit by a car, driving backwards into a garage. It was unclear if the driver drove backwards once or twice, which would indicate that he willingly injured and killed the woman. With this work, we demonstrate how 3D documentation, data merging and animation enable to answer reconstructive questions regarding the dynamic development of patterned injuries, and how this leads to a real data based reconstruction of the course of event.

Geometric facial comparisons in speed-check photographs

In many cases, it is not possible to call the motorists to account for their considerable excess in speeding, because they deny being the driver on the speed-check photograph. An anthropological comparison of facial features using a photo-to-photo comparison can be very difficult depending on the quality of the photographs. One difficulty of that analysis method is that the comparison photographs of the presumed driver are taken with a different camera or camera lens and from a different angle than for the speed-check photo. To take a comparison photograph with exactly the same camera setup is almost impossible. Therefore, only an imprecise comparison of the individual facial features is possible. The geometry and position of each facial feature, for example the distances between the eyes or the positions of the ears, etc., cannot be taken into consideration. We applied a new method using 3D laser scanning, optical surface digitalization, and photogrammetric calculation of the speed-check photo, which enables a geometric comparison. Thus, the influence of the focal length and the distortion of the objective lens are eliminated and the precise position and the viewing direction of the speed-check camera are calculated. Even in cases of low-quality images or when the face of the driver is partly hidden, good results are delivered using this method. This new method, Geometric Comparison, is evaluated and validated in a prepared study which is described in this article.

3D bloodstain pattern analysis: ballistic reconstruction of the trajectories of blood drops and determination of the centres of origin of the bloodstains

For crime scene investigation in cases of homicide, the pattern of bloodstains at the incident site is of critical importance. The morphology of the bloodstain pattern serves to determine the approximate blood source locations, the minimum number of blows and the positioning of the victim. In the present work, the benefits of the three-dimensional bloodstain pattern analysis, including the ballistic approximation of the trajectories of the blood drops, will be demonstrated using two illustrative cases. The crime scenes were documented in 3D, using the non-contact methods digital photogrammetry, tachymetry and laser scanning. Accurate, true-to-scale 3D models of the crime scenes, including the bloodstain pattern and the traces, were created. For the determination of the areas of origin of the bloodstain pattern, the trajectories of up to 200 well-defined bloodstains were analysed in CAD and photogrammetry software. The ballistic determination of the trajectories was performed using ballistics software. The advantages of this method are the short preparation time on site, the non-contact measurement of the bloodstains and the high accuracy of the bloodstain analysis. It should be expected that this method delivers accurate results regarding the number and position of the areas of origin of bloodstains, in particular the vertical component is determined more precisely than using conventional methods. In both cases relevant forensic conclusions regarding the course of events were enabled by the ballistic bloodstain pattern analysis.

Application of 3D documentation and geometric reconstruction methods in traffic accident analysis: with high resolution surface scanning, radiological MSCT/MRI scanning and real data based animation

The examination of traffic accidents is daily routine in forensic medicine. An important question in the analysis of the victims of traffic accidents, for example in collisions between motor vehicles and pedestrians or cyclists, is the situation of the impact. Apart from forensic medical examinations (external examination and autopsy), three-dimensional technologies and methods are gaining importance in forensic investigations. Besides the post-mortem multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI) for the documentation and analysis of internal findings, highly precise 3D surface scanning is employed for the documentation of the external body findings and of injury-inflicting instruments. The correlation of injuries of the body to the injury-inflicting object and the accident mechanism are of great importance. The applied methods include documentation of the external and internal body and the involved vehicles and inflicting tools as well as the analysis of the acquired data. The body surface and the accident vehicles with their damages were digitized by 3D surface scanning. For the internal findings of the body, post-mortem MSCT and MRI were used. The analysis included the processing of the obtained data to 3D models, determination of the driving direction of the vehicle, correlation of injuries to the vehicle damages, geometric determination of the impact situation and evaluation of further findings of the accident. In the following article, the benefits of the 3D documentation and computer-assisted, drawn-to-scale 3D comparisons of the relevant injuries with the damages to the vehicle in the analysis of the course of accidents, especially with regard to the impact situation, are shown on two examined cases.

A clinically applicable laser-based image-guided system for laparoscopic liver procedures

PURPOSE Laser range scanners (LRS) allow performing a surface scan without physical contact with the organ, yielding higher registration accuracy for image-guided surgery (IGS) systems. However, the use of LRS-based registration in laparoscopic liver surgery is still limited because current solutions are composed of expensive and bulky equipment which can hardly be integrated in a surgical scenario. METHODS In this work, we present a novel LRS-based IGS system for laparoscopic liver procedures. A triangulation process is formulated to compute the 3D coordinates of laser points by using the existing IGS system tracking devices. This allows the use of a compact and cost-effective LRS and therefore facilitates the integration into the laparoscopic setup. The 3D laser points are then reconstructed into a surface to register to the preoperative liver model using a multi-level registration process. RESULTS Experimental results show that the proposed system provides submillimeter scanning precision and accuracy comparable to those reported in the literature. Further quantitative analysis shows that the proposed system is able to achieve a patient-to-image registration accuracy, described as target registration error, of [Formula: see text]. CONCLUSIONS We believe that the presented approach will lead to a faster integration of LRS-based registration techniques in the surgical environment. Further studies will focus on optimizing scanning time and on the respiratory motion compensation.

Confocal imaging protocols for live/dead staining in three-dimensional carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.

Reconstruction and 3D visualisation based on objective real 3D based documentation

Reconstructions based directly upon forensic evidence alone are called primary information. Historically this consists of documentation of findings by verbal protocols, photographs and other visual means. Currently modern imaging techniques such as 3D surface scanning and radiological methods (Computer Tomography, Magnetic Resonance Imaging) are also applied. Secondary interpretation is based on facts and the examiner's experience. Usually such reconstructive expertises are given in written form, and are often enhanced by sketches. However, narrative interpretations can, especially in complex courses of action, be difficult to present and can be misunderstood. In this report we demonstrate the use of graphic reconstruction of secondary interpretation with supporting pictorial evidence, applying digital visualisation (using 'Poser') or scientific animation (using '3D Studio Max', 'Maya') and present methods of clearly distinguishing between factual documentation and examiners' interpretation based on three cases. The first case involved a pedestrian who was initially struck by a car on a motorway and was then run over by a second car. The second case involved a suicidal gunshot to the head with a rifle, in which the trigger was pushed with a rod. The third case dealt with a collision between two motorcycles. Pictorial reconstruction of the secondary interpretation of these cases has several advantages. The images enable an immediate overview, give rise to enhanced clarity, and compel the examiner to look at all details if he or she is to create a complete image.

Suitability of Common Collagen Scaffolds for Anterior Cruciate Ligament Repair

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in Ø and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore Ø 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells (MSCs) and/or tenocytes (TCs) with regards to their suitability for anterior cruciate ligament (ACL) repair. METHODS: Dynamic Intraligamentary Stabilization (DIS) utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide® (CG) and Novocart® (NC). Cells were seeded onto the scaffolds and cultured for 7 days either as a pure populations or as “premix” containing a 1 : 1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts (0.4µm). We analyzed the patches by real time polymerase chain reaction (RT-PCR), glycosaminoglycan (GAG), DNA and hydroxy-proline (HYP) content, was determined. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e. confocal laser scanning microscopy (cLSM) and scanning electron microscopy (SEM), were applied. RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and cLSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitative polymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 days. CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall

The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

Fluorescent-magnetic hybrid nanoparticles induce a dose-dependent increase in proinflammatory response in lung cells in vitro correlated with intracellular localization

Iron-platinum nanoparticles embedded in a poly(methacrylic acid) (PMA) polymer shell and fluorescently labeled with the dye ATTO 590 (FePt-PMA-ATTO-2%) are investigated in terms of their intracellular localization in lung cells and potential to induce a proinflammatory response dependent on concentration and incubation time. A gold core coated with the same polymer shell (Au-PMA-ATTO-2%) is also included. Using laser scanning and electron microscopy techniques, it is shown that the FePt-PMA-ATTO-2% particles penetrate all three types of cell investigated but to a higher extent in macrophages and dendritic cells than epithelial cells. In both cell types of the defense system but not in epithelial cells, a particle-dose-dependent increase of the cytokine tumor necrosis factor alpha (TNFalpha) is found. By comparing the different nanoparticles and the mere polymer shell, it is shown that the cores combined with the shells are responsible for the induction of proinflammatory effects and not the shells alone. It is concluded that the uptake behavior and the proinflammatory response upon particle exposure are dependent on the time, cell type, and cell culture.