Optical Imaging

Optical Coherence Tomography (OCT)We describe the fundamental concept of optical coherence tomography (OCT) and discuss the two main working principles time domain OCT and frequency domain OCT. Then, we review extended functionalities including spectrally and polarization-resolved OCT as well as Doppler-OCT and show concepts for contrast enhancement. Based on these fundamentals, we demonstrate the potential of OCT for small animal imaging on the basis of exemplary studies on retinal imaging and lung imaging.Optoacoustic ImagingThis chapter deals with the fascinating topic of optoacoustic imaging, a recent powerful addition to the arsenal of in vivo functional and molecular small animal imaging. Due to its hybrid nature, involving optical excitation and ultrasonic detection, optoacoustics overcomes the imaging depth limitations of optical microscopy related to light scattering in living tissues while further benefiting from the compelling advantages of optical contrast. To this end, optoacoustic imaging has been shown capable of delivering multiple types of imaging contrast (structural, functional, kinetic, molecular) within a single imaging modality. It can further deliver images with high spatiotemporal resolution that rivals performance of other well-established whole-body imaging modalities. As such, optoacoustics can play a vital role in biomedical research, from early disease detection and monitoring of dynamic phenomena noninvasively to accelerating drug discovery.Optical ProbesThis chapter is devoted to the properties and application of fluorescence dyes as probes for optical imaging. A variety of agents have been described to date, including nontargeting dyes, vascular agents, targeted conjugates, activatable dyes, and sensing probes. The major classes encompass polymethine dyes and xanthenes dyes, both of which are commercially available in broad variations. Addressing the purpose of optical animal imaging, the most relevant parameters to apply such probes are discussed, thereby supporting the reader in choosing reasonable imaging probes and in preparing bioconjugates for his studies

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE(-/-)mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3(+) T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3(+) T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.Peer reviewe

Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice

IntroductionThe transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a “pattern regulatory function,” by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo.Methods and resultsEndothelial Adam10 deficiency (Adam10ecko) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes.DiscussionCollectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases

Experience of running PIAF on the CS-2 at CERN

. A 32-node Meiko CS-2 MIMD machine has been installed at CERN as part of an ESPRIT project funded by the European Community. It is intended that this machine be used by CERN to run codes developed at CERN and elsewhere and to thereby demonstrate the feasibility of using a CS-2 in a demanding high energy physics environment. One part of the project deals with interactive data analysis using PIAF (Parallel Interactive Analysis Facility), a system developed at CERN for carrying out queries on very large databases of several gigabytes in parallel [1]. 1 PIAF software The PIAF system is based on the PAW (Physics Analysis Workstation) data analysis and visualization package also developed at CERN [2]. Whereas PAW is basically intended to run stand-alone on a variety of platforms ranging from small microcomputers to mainframes, PIAF is designed to interface with PAW to give the user transparent access to high-performance computing facilities which are physically located elsewhere. ..

Oxygen and nutrient delivery in tissue engineering:Approaches to graft vascularization

The field of tissue engineering is making great strides in developing replacement tissue grafts for clinical use, marked by the rapid development of novel biomaterials, their improved integration with cells, better-directed growth and differentiation of cells, and improved three-dimensional tissue mass culturing. One major obstacle that remains, however, is the lack of graft vascularization, which in turn renders many grafts to fail upon clinical application. With that, graft vascularization has turned into one of the holy grails of tissue engineering, and for the majority of tissues, it will be imperative to achieve adequate vascularization if tissue graft implantation is to succeed. Many different approaches have been developed to induce or augment graft vascularization, both in vitro and in vivo. In this review, we highlight the importance of vascularization in tissue engineering and outline various approaches inspired by both biology and engineering to achieve and augment graft vascularization

Methodological approaches in aggregate formation and microscopic analysis to assess pseudoislet morphology and cellular interactions

Microscopy has revolutionised our view on biology and has been vital for many discoveries since its invention around 200 years ago. Recent developments in cell biology have led to a strong interest in generating spheroids and organoids that better represent tissue. However, the current challenge faced by many researchers is the culture and analysis of these three-dimensional (3D) cell cultures. With the technological improvements in reconstructing volumetric datasets by optical sections, it is possible to quantify cells, their spatial arrangement, and the protein distribution without destroying the physical organization. We assessed three different microwell culture plates and four analysis tools for 3D imaging data for their applicability for the analysis of 3D cultures. A key advantage of microwell plates is their potential to perform high-throughput experiments in which cell cultures are generated and analysed in one single system. However, it was shown that this potential could be impacted by the material composition and microwell structure. For example, antibody staining was not possible in a hydrogel microwell, and truncated pyramid-structured microwells had increased background fluorescence due to their structure. Regarding analysis tools, four different software, namely CellProfiler, Fiji/ImageJ, Nikon GA3 and Imaris, were compared for their accuracy and applicability in analysing datasets from 3D cultures. The results showed that the open-access software, CellProfiler and Fiji, could quantify nuclei and cells, yet with varying results compared to manual counting, and may require post-processing optimisation. On the other hand, the GA3 and Imaris software packages showed excellent versatility in usage and accuracy in the quantification of nuclei and cells, and could classify cell localisation. Together these results provide critical considerations for microscopic imaging and analysis of 3D cell cultures