Search

Preclinical studies usually require high levels of morphological, functional, and biochemical information at subcellular resolution. This type of information cannot be obtained from clinical imaging techniques, such as MRI, PET/CT, or US. Luckily, many microscopy techniques exist that can offer this information, also for malignant tissues and therapeutic approaches. In this overview, we discuss the various advanced optical microscopy techniques and their applications in oncological research. After a short introduction in Sect. 16.1, we continue in Sect. 16.2 with a discussion on fluorescent labelling strategies, followed in Sect. 16.3 by an in-depth description of confocal, light-sheet, two-photon, and super-resolution microscopy. We end in Sect. 16.4 with a focus on the applications, specifically in oncology.

Parameters such as pretreatment method, enzyme type and concentration, determine the conversion efficiency of biomass' cellulose and hemicellulose to glucose and mainly xylose in biomass-based fuel production. Chemical quantification of these processes offers no information on the effect of enzymatic hydrolysis (EH) on particle morphology. We report on the development of a microscopy method for imaging pretreated biomass particles at different EH stages. The method was based on acquiring large field of view images, typically 20×10 mm2 containing thousands of particles. Morphology of particles with lengths between 2 μm and 5 mm could be visualized and analyzed. The particle length distribution of corn stover samples, pretreated with increasing amounts of sulfuric acid at different EH stages, was measured. Particle size was shown to be dependent on pretreatment severity and EH time. The methodology developed could offer an alternative method for characterization of EH of biomass for second generation biofuels and visualization of recalcitrant structures.

Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy—imaging the sample from different angles followed by 3D image reconstruction—was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures. [ABSTRACT FROM AUTHOR]

Purpose We developed 2-photon laser scanning microscopy analysis of the native murine bladder. Materials and Methods Bladder tissue from wild-type mice was imaged by 2-photon laser scanning microscopy autofluorescence and second harmonic generation microscopy. Bladder wall layers and structures were analyzed using differences in color, size, shape and morphology. Results Autofluorescence of the urothelium, nerve structures and muscles was visible in the green spectral channel due to autofluorochromes such as NAD(P)H and elastin. Second harmonic generation of collagen was seen in the blue spectral channel. Imaging from the mucosal side revealed umbrella cells at 0 and 30 μm, of which the high cellular NAD(P)H content allows autofluorescence detection. Below that a network-like connective tissue layer was visualized up to 50 μm that contained vessels with a diameter of 10 to 40 μm and nerves with a diameter of 1 to 6 μm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 μm. Below at 20 to 25 μm we visualized a thick muscle layer containing elastic fibers and macrophages. Findings were also represented in 3-dimensional reconstructions, providing information on structure localization, orientation and interconnection. Conclusions Two-photon laser scanning microscopy imaging using autofluorescence of the murine bladder is a promising technique to provide new insight into structures and morphology. It opens avenues to identify structural changes in bladder pathology. [ABSTRACT FROM AUTHOR]

Background: The endothelial glycocalyx (EG) is the carbohydrate-rich luminal lining of endothelial cells that mediates permeability and blood cell-vessel wall interactions. To establish an atheroprotective role of the EG, adequate imaging and quantification of its properties in intact, viable, atherogenesis-prone arteries is needed. Methods: Carotid arteries of C57Bl6/J mice (n = 22) were isolated including the bifurcation, mounted in a perfusion chamber, and perfused with fluorescent lectin wheat germ agglutinin-fluorescein isothiocyanate. The EG was visualized through the vessel wall using two-photon laser scanning microscopy. An image quantification protocol was developed to assess EG thickness, which was sensitive to hyaluronidase-induced changes. Results: In the lesion-protected common carotid artery, EG thickness was found to be 2.3 ± 0.1 μm (mean ± SEM), while the surface area devoid of (wheat germ agglutinin-sensitive) EG was 8.9 ± 4.2%. Data from the external carotid artery were similar (2.5 ± 0.1 μm; 9.1 ± 5.0%). In the atherogenesis-prone internal carotid artery the EG-devoid surface area was significantly higher (27.4 ± 5.5%, p < 0.05); thickness at the remaining areas was 2.5 ± 0.1 μm. Conclusion: The EG can be adequately imaged and quantified using two-photon laser scanning microscopy in intact, viable mounted carotid arteries. Spatial EG differences could underlie atherogenesis. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] Intravital microscopy (IVM) expands our understanding of cellular and molecular processes, with applications ranging from fundamental biology to (patho)physiology and immunology, as well as from drug delivery to drug processing and drug efficacy testing. In this review, we highlight modalities, methods and model organisms that make up today's IVM landscape, and we present how IVM - via its high spatiotemporal resolution - enables analysis of metabolites, small molecules, nanoparticles, immune cells, and the (tumor) tissue microenvironment. We furthermore present examples of how IVM facilitates the elucidation of nanomedicine kinetics and targeting mechanisms, as well as of biological processes such as immune cell death, host-pathogen interactions, metabolic states, and disease progression. We conclude by discussing the prospects of IVM clinical translation and examining the integration of machine learning in future IVM practice. [ABSTRACT FROM AUTHOR]