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Deep Tissue Imaging

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  • Multiphoton Microscopy – a Satisfied Wish List

    The colorful picture shows colon tumor cells, fluorescently labelled and lineage traced from a multicolor tracer. The gray color codes for the second harmonic generation (SHG) signal from Collagen 1. Lineage traced tumor cells are shown in magenta, blue, green, yellow and red. All channels were recorded with two-photon excitation, using the SP8 DIVE by Leica Microsystems. Sample and image were kindly provided by J. van Rheenen, H. Snippert, Utrecht (the Nederlands,) and I. Steinmetz, Leica Microsystems Mannheim.
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  • Five Questions Asked: Prof. Dr. Jacco van Rheenen speaks about the most important considerations when imaging deep into mouse tissue

    When operating a confocal microscope, or when discussing features and parameters of such a device, we inescapably mention the pinhole and its diameter. This short introductory document is meant to explain the significance of the pinhole for those, who did not want to spend too much time to dig into theory and details of confocal microscopy but wanted to have an idea about the effect of the pinhole.
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  • Multiphoton Microscopy Publication List

    Multiphoton Microscopy is an advanced technique for imaging thick samples. Applications range from the visualization of the complex architecture of the whole brain to the study of tumor development and metastasis or the responses of the immune system in living animals. On this regularly updated reference list you can find selected publications on reseach using multiphoton microscopy.
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  • BABB Clearing and Imaging for High Resolution Confocal Microscopy: Counting and Sizing Kidney Cells in the 21st Century

    Multipohoton microscopy experiment using Leica TCS SP8 MP and Leica 20x/0.95 NA BABB immersion objective. Understanding kidney microanatomy is key to detecting and identifying early events in kidney disease. Improvements in tissue clearing and imaging have been crucial in this field, and now we report on a novel, time-efficient method to study podocyte depletion in renal glomeruli using a combination of immunofluorescence, optical clearing, confocal microscopy and 3D analysis.
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  • Clearing of Fixed Tissue: A Review from a Microscopist’s Perspective

    Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples.
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  • Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent

    Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations.
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  • Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.
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  • Deeper Insights in Transparent Animals

    CLARITY clearing derivatives for multiphoton microscopy. Transparent organisms help us to identify spatial arrangements and connections of cells and tissues, especially neuronal circuits can easily be identified and characterized. CLARITY is on everyone's lips.
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  • Clearing Procedures for Deep Tissue Imaging

    Multi-channel multiphoton microscopy with dedicated optics for CLARITY. Why clearing? Curiosity is human nature. And nothing attracts as much curiosity as the inside of living organisms. While in ancient times those who cut human bodies open to do research were put to death, and modern anatomy started only after Pope Clement VII allowed dissection, we can now watch brains working in living animals – and have a good chance of soon being able to interfere with the observed activities for healing (or control) purposes.
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  • Map the Brain with CLARITY

    Imaging whole brains with CLARITY and multiphoton microscopy. Image a whole brain without sectioning? Investigate neuronal circuits without reconstruction? Perform molecular phenotyping without destroying subcellular structures? Understanding the brain with molecular resolution and global scope has always been challenging. The novel CLARITY method, developed by the Deisseroth laboratory at Stanford University, USA, pushes the barrier of deep tissue imaging a big step ahead.
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  • Structural and Molecular Interrogation of Intact Biological Systems

    To understand structure and function of brains or other complex biological systems, the method of choice is microscopy. In particular, confocal microscopy is employed to reveal three-dimensional connectivity and functional interactions. To come to a real insight into brain’s way of working, one must look deep into the tissue – which usually is non-transparent. A couple of clearing methods have been developed in the past, but they usually come along with distortions of the structures, incompatibilities with fluorescence stainings or are just prohibitively toxic to the lab technician.
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  • Light Sheet Fluorescence Microscopy: Beyond the Flatlands

    Light Sheet Fluorescence Microscopy (LISH-M) is a true fluorescence optical sectioning technique, first described by Heinrich Siedentopf in 1902 under the name of Ultramicroscopy. Light sheet microscopy utilises a plane of light to optically section samples. This allows deep imaging within transparent tissues and whole organisms. This book chapter will provide the reader with a comprehensive view on this emerging technology.
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  • Principles of Multiphoton Microscopy for Deep Tissue Imaging

    Basics of multiphoton microscopy. This interactive tutorial explains the principles of multiphoton microscopy for deep tissue imaging. Multiphoton microscopy uses excitation wavelengths in the infrared taking advantage of the reduced scattering of longer wavelengths. This makes multiphoton imaging the perfect tool for deep tissue imaging in thick sections and living animals.
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  • Light Sheet Fluorescence Microscopy - A Review

    Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy.
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  • Webinar: The Solution for Deep Imaging

    Imaging of thick specimen using multiphoton microscopy. Multiphoton microscopy is the method of choice for non-invasive deep-penetration fluorescence microscopy of thick highly scattering samples. Good results have already been obtained with a diversity of specimen, e.g. lymphatic organs, kidney, heart, skin and brain (slices as well as whole organs, fixed specimen as well as living cells).
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  • New Standard in Electrophysiology and Deep Tissue Imaging

    The function of nerve and muscle cells relies on ionic currents flowing through ion channels. These ion channels play a major role in cell physiology. One way to investigate ion channels is to use patch clamping. This method allows investigation of ion channels in detail and recording of the electric activity of different types of cells, mainly excitable cells like neurons, muscle fibres or beta cells of the pancreas. The patch clamping technique was developed by Erwin Neher and Bert Sakmann in the 1970s and 80s to study individual ion channels in living cells. In 1991 they received the Nobel Prize for Physiology and Medicine for their work. Today the patch clamping technique is one of the most important methods in the field of electrophysiology.
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