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Imaging Keratan Sulfate in Ocular Tissue Sections by Immunofluorescence Microscopy and LA-ICP-MS
(2022)
Carbohydrate-specific antibodies can serve as valuable tools to monitor alterations in the extracellular matrix resulting from pathologies. Here, the keratan sulfate-specific monoclonal antibody MZ15 was characterized in more detail by immunofluorescence microscopy as well as laser ablation ICP-MS using tissue cryosections and paraffin-embedded samples. Pretreatment with keratanase II prevented staining of samples and therefore demonstrated efficient enzymatic keratan sulfate degradation. Random fluorescent labeling and site-directed introduction of a metal cage into MZ15 were successful and allowed for a highly sensitive detection of the keratan sulfate landscape in the corneal stroma from rats and human tissue.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is nowadays a versatile and powerful analytical method for direct solid sample analysis. The applicability has been demonstrated for a wide variety of samples covering hard and soft materials. In an imaging mode the technique provides quantitative information on the elemental distribution within a sample. LA-ICP-MS imaging is of particular interest in biomedical research as the distribution of an element gives valuable insight on uptake and distribution of essential and toxic trace elements, administered contrast agents as well es nanoparticles. LA-ICP-MS is therefore a powerful complement to other imaging techniques. Recent instrumental improvements, especially in sample chamber design, have contributed to better sensitivity and spatial resolution enabling subcellular imaging. The book chapter provides a comprehensive overview about spatially resolved localisation and quantification of various nanoparticles in cells and tissue thin sections by LA-ICP-MS. Furthermore, different sample preparation strategies and internal standardisation and calibration approaches for bioimaging by LA-ICP-MS are summarized and discussed.
Metal-containing nanomaterials are used in numerous fields ranging from industrial applications to nanomedicine. Several studies have demonstrated that the physicochemical properties of nanoparticles have an impact on their pharmacokinetics, transfer and clearance. The high sensitivity and multielement capability of LA-ICP-MS enables the elucidation of interactions between tissue components and nanomaterials used as imaging probes or drug carriers. Potential toxic effects are investigated as well. Thus, LA imaging significantly supports the clinical translation of safe and efficient nanoparticles for diagnostic and therapeutic purposes.
In a standard high magnification two-photon microscopy (2PM) setup the scan is achieved by moving the excitation beam inside the beam path limits of the microscope. Thus, the scanning area of the focus is limited by the field of view of the objective lens. As a consequence, the maximum scanning area is optically restricted to a diameter of 0.5 to 0.7 mm which impairs the application in biological and biomedical research.
Herein, wide-field 2PM was used to image every dimension of the human hair follicle in whole tissue samples in situ. Therefore, a 2P microscope with a new approach called 2PM FlySCAN was applied. The 2P microscope moves the scan lens in a flying optics setup: The focus of the excitation laser beam is always concentric and collinear with the scan lens. The laser focus is in a fixed axial position to the scan lens. This approach allows to scan a large field of view (7×10 mm2) up to a resolution of 500 nm2 and a scan depth ~ 500 µm without vignetting or distortion.
Further, a new en bloc longitudinal imaging mode was implemented which allows to image hair follicles in full length at a glance in a histomorphological correlation without further processing or sectioning. In situ labelling was successfully applied to investigate certain cell populations in the hair follicle which is of highly clinical relevance for various issues.
Our results demonstrate that the here used wide-field 2P microscope combined with the en bloc longitudinal imaging mode is a promising approach for the preclinical assessment of the human hair follicle addressing the demand for an appropriate sample preparation and analysis techniques.