Two-Photon Endoscopy: State of the Art and Perspectives

In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications

Two-Photon Endoscopy: State of the Art and Perspectives

In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G > T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.</p

Two-photon microscopy and its applications in (micro) vascular research

Currently many thermoregulatory models incorporate cutaneous perfusion, as it plays a major role in heat transport. The magnitude of perfusion is generally described as a function of temperature. Some physiological models, such as formulated by Stolwijk, Tanabe and Fiala, incorporate central tonus and local sensitivity explicitly. However, used perfusion control parameters have been estimated and are not based on physiological measurements. Therefore, their applicability might not be justified. In this research a method is proposed to measure central sympathetic vasoconstrictor tone and local cutaneous vasoconstrictor sensitivity. Together, the protocol was performed on males and females, young adults (18-28yr) and elderly (68-78yr) and on both glabrous and non-glabrous skin. Local cutaneous perfusion is measured by laser-Doppler flowmetry. Through iontophoresis of Bretylium tosylate (10mM) sympathetic nerve terminals are blocked, such that local endogenous release of sympathetic neurotransmitters is abolished. Afterwards a superphysiological dose noradrenaline (2.5mM) is administered through iontophoresis. The reduction in perfusion is used as a measure for vasoconstrictor sensitivity at maximal vasoconstrictor tone. This is repeated over a range of local temperatures. Then the whole body is cooled and the decrease in perfusion at a non-bretylium site is measured. Combining perfusion and local sensitivity, vasoconstrictor tone is described as a function of temperature