Dual-Objective Pointillism Microscopy Setup with Interferometric and Astigmatic Detection
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In the past decade, imaging has advanced to become a crucial tool in fundamental and biomedical research and it has become increasingly important to be able to image whole organs with single cell resolution. Light sheet fluorescence microscopy, also called selective plane illumination microscopy or ultramicroscopy, provides a high resolution in transparent and intact whole organs. By the application of a thin light sheet, only a defined slice of the specimen is illuminated and the fluorescence signal is detected by an objective perpendicular to the specimen. By moving the specimen vertically through the laser, a z-stack is acquired which corresponds to an optical sectioning without physical disruption of the specimen. The data can further be reconstructed to a three-dimensional volume and analysed in its entire complexity in micrometre resolution. This article reviews the prerequisites for successful light sheet fluorescence microscopy, in terms of tissue preparation and optical clearing, and highlights recent advances and applications in the context of basic and biomedical research, with special focus on the central nervous system of rodents.
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A major limitation of any type of microscope is the penetration depth in turbid tissue.Here, we demonstrate a fundamentally novel kind of fluorescence microscope that images through optically thick turbid layers.The microscope uses scattered light, rather than light propagating along a straight path, for imaging with subwavelength resolution.Our method uses constructive interference to focus scattered laser light through the turbid layer.Microscopic fluorescent structures behind the layer were imaged by raster scanning the focus.
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Selective plane illumination microscopy (SPIM) and other fluorescence microscopy techniques in which a focused sheet of light serves to illuminate the sample have become increasingly popular in developmental studies. Fluorescence light-sheet microscopy bridges the gap in image quality between fluorescence stereomicroscopy and high-resolution imaging of fixed tissue sections. In addition, high depth penetration, low bleaching and high acquisition speeds make light-sheet microscopy ideally suited for extended time-lapse experiments in live embryos. This review compares the benefits and challenges of light-sheet microscopy with established fluorescence microscopy techniques such as confocal microscopy and discusses the different implementations and applications of this easily adaptable technology.
Photoactivated localization microscopy
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Preface.Fundamentals of Light Microscopy Light and Color Illuminators, Filters, and Isolation of Specific Wavelengths Lenses and Geometrical Optics Diffraction and Interference in Image Formation Diffraction and Spatial Resolution Phase Contrast Microscopy and Dark - Field Microscopy Properties of Polarized Light Polarization Microscopy Differential Interference Contrast (DIC) Microscopy and Modulation Contrast Microscopy Fluorescence Microscopy Confocal Laser Scanning Microscopy Video Microscopy Digital CCD Microscopy Digital Image Processing Image Processing for Scientific Publication.Appendix I.Appendix II.Appendix III.Glossary.References.Index.
Digital Holographic Microscopy
Dark field microscopy
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Polarized light microscopy
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A modified tilted perpendicular medium has been proposed. With crystalline easy axes oriented randomly at ±45° relative to the perpendicular direction in the cross-track plane, the in-plane magnetic charges in the uniformly tilted perpendicular media can be effectively eliminated. Recording properties of the tilted perpendicular media are studied via micromagnetic simulation. In addition to the advantage of less saturation field, titled perpendicular media also show improved recording performance as compared to the conventional perpendicular media.
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We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three-dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.
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Two-photon excitation microscopy
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Interference microscopy
Digital Holographic Microscopy
Polarization Microscopy
Polarized light microscopy
Dark field microscopy
Bright-field microscopy
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We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three-dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.
Interference microscopy
Optical sectioning
Bright-field microscopy
Two-photon excitation microscopy
Photoactivated localization microscopy
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This chapter focuses on the basics of the light sheet microscopy principle and construction, followed by an overview of its various implementations. It discusses some of the novel data handling solutions that light sheet microscopy gave rise to, as well as future prospects in this area. Two different ways of generating a light sheet are commonly used: in selective plane illumination microscopy (SPIM), the laser beam is expanded and focused using a cylindrical lens to form the sheet of light. Alternatively, a "virtual" light sheet can be generated by the digitally scanned light sheet microscopy (DSLM) technique, which uses a scanning mirror to rapidly sweep a beam across the field of view (FOV). The chapter discusses how to acquire 2D images of a sample using light sheet microscopy. However, most biological applications require 3D imaging. Given its excellent optical sectioning capability, speed, and low phototoxicity, light sheet microscopy is ideally suited for fast 3D imaging.
Optical sectioning
Bright-field microscopy
Structured Light
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