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What is Confocal Laser Scanning Microscopy?

Confocal Laser Scanning Microscopy (CLSM) is one of a series of methods to generate slices from microscopic samples by means of optics. The sample stays intact, and the slicing may be repeated many times. True Confocal Scanning (TCS) is a technique, where only a single, diffraction limited spot is illuminated and observed at a time. The benefit of confocal imaging is a dramatically increased contrast by removal of out-of-focus haze. Z-sequences of optical slices (3D image stacks) are sources for subsequent rendering as anaglyphes, depth-coded maps or 3D movies. TCS is also very well compatible with multi-fluorescence imaging, time-lapse imaging, FLIM, FRAP and FCS measurements – plus a whole world of spectral applications.

  • Axial Tubule Junctions Control Rapid Calcium Signaling in Atria

    The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface.
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  • Primary Beam Splitting Devices for Confocal Microscopes

    Current fluorescence microscopy employs incident illumination which requires separation of illumination and emission light. The classical device performing this separation is a color-dependent beam splitting mirror which has fixed spectral parameters and transmits the emission usually between 90% and 98% within the designated bands. Transmission is wavelength dependent and also differs by technology, requirements and design. An alternative is the acousto optical beam splitter which has freely tunable reflection notches and transmits the emission on average at 95% between these notches.
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  • Pinhole Effect in Confocal Microscopes

    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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  • Human NK Cell Development Requires CD56-mediated Motility and Formation of the Developmental Synapse

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo.
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  • Testing the Münch Hypothesis of Long Distance Phloem Transport in Plants

    Long distance transport in plants occurs in sieve tubes of the phloem. The pressure flow hypothesis introduced by Ernst Münch in 1930 describes a mechanism of osmotically generated pressure differentials that are supposed to drive the movement of sugars and other solutes in the phloem, but this hypothesis has long faced major challenges. The key issue is whether the conductance of sieve tubes, including sieve plate pores, is sufficient to allow pressure flow. We show that with increasing distance between source and sink, sieve tube conductivity and turgor increases dramatically in Ipomoea nil. Our results provide strong support for the Münch hypothesis, while providing new tools for the investigation of one of the least understood plant tissues.
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  • Clarifying Tissue Clearing

    Biological specimens are intrinsically three dimensional; however because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This review introduces the physical basis for light-scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.
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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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  • Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index

    Application example of HyVolution Super-Resolution - Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high-end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented.
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  • Multispectral Phloem-Mobile Probes: Properties and Applications

    Using Arabidopsis (Arabidopsis thaliana) seedlings, we identified a range of small fluorescent probes that entered the translocation stream and were unloaded at the root tip. These probes had absorbance/emission maxima ranging from 367/454 to 546/576 nm and represent a versatile toolbox for studying phloem transport. Of the probes that we tested, naturally occurring fluorescent coumarin glucosides (esculin and fraxin) were phloem loaded and transported in oocytes by the sucrose transporter, AtSUC2. Arabidopsis plants in which AtSUC2 was replaced with barley (Hordeum vulgare) sucrose transporter (HvSUT1), which does not transport esculin in oocytes, failed to load esculin into the phloem.
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  • P53- and Mevalonate Pathway–Driven Malignancies Require Arf6 for Metastasis and Drug Resistance

    Application example of HvYolution Super-Resolution - Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial–mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program.
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  • C. Elegans

    Work Efficiently in Developmental Biology with Stereo and Confocal Microscopy: C. elegans

    For scientists, technicians, and teachers working with the worm C. elegans in the research lab or classroom, this report is intended to give useful information to help improve their daly work. The aim is to make the work steps of worm picking, transgenesis, RNA interference, screening, and functional imaging efficient. It also details the various possibilities for equipping a research worm lab or biology classroom/teaching lab explaining worm methods.
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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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  • Adeno-associated Viral Vectors do not Efficiently Target Muscle Satellite Cells

    Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes, and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to postmitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice.
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  • Highly Selective Fluorescent and Colorimetric Probe for Live-cell Monitoring of Sulphide Based on Bioorthogonal Reaction

    H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety.
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  • Label-free in vivo Imaging of Myelinated Axons in Health and Disease with Spectral Confocal Reflectance Microscopy

    We report a new technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths.
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  • The Bimodally Expressed MicroRNA miR‐142 Gates Exit from Pluripotency

    A stem cell's decision to self‐renew or differentiate is thought to critically depend on signaling cues provided by its environment. It is unclear whether stem cells have the intrinsic capacity to control their responsiveness to environmental signals that can be fluctuating and noisy. Using a novel single‐cell microRNA activity reporter, we show that miR‐142 is bimodally expressed in embryonic stem cells, creating two states indistinguishable by pluripotency markers.
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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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  • HyVolution – Super-Resolution Imaging with a Confocal Microscope

    Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. In this article we describe the HyVolution concept and how the combination of confocal multiparameter fluorescence imaging at the confocal super-resolution regime with psf-based real deconvolution allows high-speed multicolor imaging with a resolution down to 140 nm.
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  • HyVolution – the Smart Path to Confocal Super-Resolution

    Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.
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  • MyScope for Global Research Training

    MyScope is an online educational site that provides modules on scanning and transmission electron microscopy, scanning probe and atomic force microscopy, confocal microscopy, X-ray diffraction and microanalysis. These advanced research techniques are important in characterization of materials and understanding biological processes. Developed by technique experts across the Australian Microscopy and Microanalysis Research Facility (AMMRF), quality and usability are assured.
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  • Light Sheet Microscopy Turned Vertically

    Living cells and organisms often suffer from the high light intensities used for fluorescent imaging. Light sheet microscopy reduces phototoxic effects and bleaching by illuminating a specimen in only a single plane at a time. A new light sheet microscope combines light sheet and confocal microscopy in one system without compromising either functionality and allows the combination of the two methods, e.g. confocal photomanipulation with subsequent light sheet acquisition, for new applications.
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  • "The Leica Digital Light Sheet Module – a Clever Example of Thinking Out of the Box"

    Bram van den Broek is a postdoctoral fellow at the Netherlands cancer institute in Amsterdam where he supports the advanced microscopy techniques in the laboratory of Kees Jalink. Working with Leica Microsystems as a collaboration partner for beta-testing of microscopes he enjoys very much.
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  • Novel Microscopy-Based Screening Method Reveals Regulators of Contact-Dependent Intercellular Transfer

    Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators.
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  • Progressive Glucose Stimulation of Islet Beta Cells Reveals a Transition From Segregated to Integrated Modular Functional Connectivity Patterns

    Collective beta cell activity in islets of Langerhans is critical for the supply of insulin within an organism. In order to get a detailed insight into the functional organization of the syncytium, we applied advanced analytical tools from the realm of complex network theory to uncover the functional connectivity pattern among cells composing the intact islet.
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  • From Light to Mind: Sensors and Measuring Techniques in Confocal Microscopy

    This article outlines the most important sensors used in confocal microscopy. By confocal microscopy, we mean "True Confocal Scanning", i.e. the technique that illuminates and measures one single point only. The aim is not to impart in-depth specialist knowledge, but to give the user a small but clear overview of the differences between the various technologies and to advise on which sensor may be most suitable for which applications.
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  • "We can go home and the imaging is done automatically by the Leica HCS A Matrix Screener."

    Jutta Maria Bulkescher is the technical coordinator in the Novo Nordisk Foundation Center for Protein Research and Danish Stem Cell Center in Copenhagen, Denmark. The Leica HCS-A matrix screener is an invaluable tool for her facility. "It just gives us the biggest and easiest flexibility we can have to set up different imaging paramters and to check different conditions on one multi-well plate", explains Bulkescher.
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  • iMSRC: Converting a Standard Automated Microscope into an Intelligent Screening Platform

    Microscopy in the context of biomedical research is demanding new tools to automatically detect and capture objects of interest. The few extant packages addressing this need, however, have enjoyed limited uptake due to complexity of use and installation. To overcome these drawbacks, we developed iMSRC, which combines ease of use and installation with high flexibility and enables applications such as rare event detection and high-resolution tissue sample screening, saving time and resources.
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  • Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

    Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope.
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  • Webinar: The Best of Both Worlds: Combining Light Sheet and Confocal Microscopy

    Living cells and organisms often suffer from high light intensities that are used in conventional imaging. Light sheet microscopy reduces phototoxic effects and bleaching, by only illuminating a specimen in a single plane at a time whilst the signal is detected in a perpendicular direction. In combination with high-speed cameras for image acquisition, light sheet microscopy is a very gentle method to observe fast biological processes in sensitive organisms over an extended time period.
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Interactive Tutorials

Useful Links

Communities and Web Sources
Social network for scientists
Confocal Microscopy Mailing List, University of Minnesota
Teaching tools, video lectures on biology and microscopy
Teaching tools, video lectures on biology and microscopy
Online magazine and community for molecular and cell biology researchers
Resource for high-end scientific illustrations, images and animations

Search Engines and Data Bases
Public resource database of images, videos, and animations of cells
Bioinformatic meta search engine for genes and proteins
Search interface for pubmed
List of academic databases and search engines
Beta of Google's search engine for scientific article abstracts

Directory of open access journals
The EMBO Journal
CBE-Life Sciences Education – an ASCB online journal
Biweekly publication of exceptional research articles
Journal of Cell Science
The Journal of Experimental Biology
DMM Disease Models & Mechanisms
International Journal of Life Science Methods
Collection of Journals and Proceedings in Optics and Photonics
SPIE - peer-reviewed journals on applied research in optics and photonics
Journal of Biophotonics
International, peer-reviewed, open-access, online publication
Proceedings B - the Royal Society's biological research journal
International Journal for microscopists

Microscopy Society of America
European Microscopy Society
Royal Microscopical Society
ASCB American Society of Cell Biology
the company of biologists

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