New Information from Large Tissue Volumes to the Smallest Structures of the Cell: What New Methods and Electron Microscopy Can Do for Your Research
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This article presents an overview of microscopy and its ability to assist in understanding what happens in cells and tissues. From the 1960s to 1980s, electron microscopy was the best way to understand cell processes, but the advent in the mid-1980s of light microscopy and the ability to do fluorescence imaging displaced electron microscopy in this area. However, the 21st century has seen several improvements in electron microscopy that, along with the need for more detailed ultrastructural information, make it again very attractive in the study of cells, tissues, and organs, and electron microscopy has resumed its place as the preeminent method in understanding cell processes.A septum-rich fraction was prepared from Triehophyton mentagrophytes, and the ultrastructural investigation of septa was carried out by shadowing and thin section electron microscopy before and after enzymatic and/or alkaline treatments. Tri-lamellar structure, consisting of an electron lucid middle layer and two outer electron dense layers, was observed by thin section electron microscopy. By shadowing electron microscopy, the surface ultrastructure of septa exhibited a randomly orientated microfibrillar network structure, which may correspond to the electron dense outer layers of the septum observed in thin section preparations. However, shadowed septa after papain digestion revealed a spiral arrangement of microfibrils consisting of rodlet-like units, which disappeared during chitinase treatment. This spiral structure may therefore be composed primarily of chitin. It was suggested that this spirally arranged microfibrils may correspond to the electron lucid middle layer observed in thin section preparations.
Thin section
Morphology
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Photoacoustic microscopy is dominantly sensitive to the endogenous optical absorption, while a fluorescence optical microscopy can detect the fluorescence emission to obtain the image of a sample. To some extent, the physical processes of the 2 methods are opposite, one is absorption and another is emission, but both can be used to image cells. In this article, a simultaneous dual-mode imaging system of photoacoustic microscopy and fluorescence optical microscopy is set up to image tobacco cells. Furthermore, gold nanoparticles, which have a large absorption coefficient and enough fluorescence emission with wavelength of 512 nm, are used to label certain drugs and added to the tobacco cells. Then based on the simultaneous dual-mode microscopy imaging system, the photoacoustic microscopy and fluorescence optical microscopy images of gold nanoparticle-labeled tobacco cells are obtained. The final purpose of this experimental research is to detect if the labeled drugs can enter the cells by the positions of the gold nanoparticles. This will help the experts to deliver organic pesticide more accurately and effectively. The experimental results show that by gold nanoparticle labeling technology, the imaging quality of photoacoustic microscopy and fluorescence optical microscopy can be improved, which indicates that the drugs probably enter the tobacco cells successfully.
Fluorescence-lifetime imaging microscopy
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Fluorescence-lifetime imaging microscopy
Fiducial marker
Photoactivated localization microscopy
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Photoactivated localization microscopy
STED microscopy
Fluorescence-lifetime imaging microscopy
Two-photon excitation microscopy
Digital Holographic Microscopy
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The authors present the results of a comparative microscopic study of 502 sputum smears from the patients of the Republican Tuberculosis Dispensary. Sputum smear microscopy with the Ziehl Neelsen (ZN) method and fluorescence microscopy (FM) using a Mikmed 2 (LOMO) microscope and an Olympus SX microscope with a LED Lumin attachment were compared. The latter allows conversion of a light microscope to a fluorescence one. For 2 months sputum was randomly sampled from patients at the moment of diagnosis and from those who were treated at a clinic. A culture study was used as the gold standard, among other things, to calculate the sensitivity and specificity of different microscopy methods. The sensitivity of ZN microscopy, FM on a Mikmed microscope, FM using the Lumin attachment was 28.5, 52.5, and 72.8%, respectively. The Lumin attachment is a inexpensive, portable device that converts practically all models of light microscopes to fluorescence ones. Its life is as long as 25 years, it requires maintenance and both the routine supply line and a Krona storage battery or similar ones, as well as a solar battery may be used as a source of energy.
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Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.
Correlative
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Preface.- Acknowledgements.- Dedication.- Ray Optics, Wave Optics and Imaging System Designing.- Basics of Electromagnetic Theory for Fluorescence Microscopy.- Electric Field Effects in Optical Microscopy Systems.- Quantum Description of Radiation Field and Optical Microscopy.- Molecular Physics of Fluorescence Markers.- Basics of Fluorescence and Photophysics.- General Fluorescence Imaging Techniques.- Multiphoton Fluorescence Microscopy.- Super Resolution Fluorescence Microscopy.- Image Reconstruction Methodologies for Fluorescence Microscopy.- Future Prospective of Fluorescence Microscopy.- Appendix I.- Appendix II.- Appendix III.
Photoactivated localization microscopy
Fluorescence-lifetime imaging microscopy
Fluorescence cross-correlation spectroscopy
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