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What is Live-Cell Imaging?

Besides the structural organization of cells or organs, dynamic processes are a major contributor to a functioning biological entity. Naturally, these processes can be best observed in living cells with non-invasive techniques like optical methods, collectively called “live-cell imaging” methods. Live-cell imaging covers all techniques where live cells are observed with microscopes – from the observation of embryogenesis with stereo microscopes, via cell growth studies with compound microscopes, until studies of physiological states of cells or cellular transport using fluorescent dyes or proteins. Although being highly demanding for both, experimenter and equipment (e.g. imaging systems, climate control), live-cell imaging techniques deliver results that are indispensable for present-day research.

  • Introduction to Mammalian Cell Culture

    Mammalian cell culture is one of the basic pillars of life sciences. Without the ability to grow cells in the lab, the fast progress in disciplines like cell biology, immunology, or cancer research would be unthinkable. This article gives an overview of mammalian cell culture systems. Mainly, they can be categorized according to their morphology, as well as cell type and organization. Moreover, you can find basic information about the correct growth conditions and what kind of microscope you need to watch your cells.
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  • Graphene-Based Microbots for Toxic Heavy Metal Removal and Recovery from Water

    Heavy metal contamination in water is a serious risk to the public health and other life forms on earth. Current research in nanotechnology is developing new nanosystems and nanomaterials for the fast and efficient removal of pollutants and heavy metals from water. Here, we report graphene oxide-based microbots (GOx-microbots) as active self-propelled systems for the capture, transfer, and removal of a heavy metal (i.e., lead) and its subsequent recovery for recycling purposes.
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  • Real Time Observation of Neutrophil White Blood Cell Recruitment to Bacterial Infection In Vivo

    The zebrafish (Danio rerio) is an emerging vertebrate model organism to study infection. The transparent larva comprises a fully functional innate immune system and enables live imaging of fluorescent immune cells in transgenic animals. Zebrafish infection models have been developed for both the human bacterial pathogen Shigella flexneri and the natural fish bacterial pathogen Mycobacterium marinum. Importantly, whilst S. flexneri causes acute infection and is typically used as an inflammatory paradigm, M. marinum causes a chronic disease similar to tuberculosis in humans. Here, we use real time fluorescence microscopy to image transgenic zebrafish larvae with neutrophils (granulocyte white blood cells) expressing the green fluorescent protein eGFP.
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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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  • Clap On, Clap Off. Protein On, Protein Off

    Unlike genetic or pharmacological manipulation, light-controlled proteins respond immediately, can be temporally and spatially triggered, are reversible, and are specific to the protein of interest. However, designing and using light-controllable proteins often requires expertise and specialized equipment. Now, two recently published articles in Science describe generalizable methods for making photo-controllable proteins.
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  • Chronic Inflammation Under the Microscope

    In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment.
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  • Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

    Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS , and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS , the STED- FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.
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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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  • Factors to Consider When Selecting a Research Microscope

    An optical microscope is often one of the central devices in a life-science research lab. It can be used for various applications which shed light on many scientific questions. Thereby the configuration and features of the microscope are crucial for its application coverage, ranging from brightfield through fluorescence microscopy to live-cell imaging. This article provides a brief overview of the relevant microscope features and wraps up the key questions one should consider when selecting a research microscope.
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  • Discovery of Novel Peptides Targeting Pro-Atherogenic Endothelium in Disturbed Flow Regions -Targeted siRNA Delivery to Pro-Atherogenic Endothelium in vivo

    Application example of HyVolution Super-Resolution - Atherosclerosis occurs preferentially in arterial regions exposed to disturbed blood flow. Targeting these pro-atherogenic regions is a potential anti-atherogenic therapeutic approach, but it has been extremely challenging. Here, using in vivo phage display approach and the partial carotid ligation model of flow-induced atherosclerosis in mouse, we identified novel peptides that specifically bind to endothelial cells (ECs) exposed to disturbed flow condition in pro-atherogenic regions.
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  • Work More Efficiently in Developmental Biology With Stereo Microscopy: Zebrafish, Medaka, and Xenopus

    Among the aquatic model organisms used in molecular and developmental biology the most prominent are the zebrafish (genus species: Danio rerio), medaka or japanese rice fish (genus species: Oryzias latipes), and african clawed frog (genus species: Xenopus laevis). This report gives useful information to scientists and technicians which can help improve their daily laboratory work by making the steps of transgenesis, fluorescent screening, and functional imaging more efficient.
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  • Work More Efficiently In Developmental Biology With Stereo Microscopy: Fruit Flies (Drosophila Melanogaster)

    For scientists and technicians working with fruit flies, most often genus Drosophila, this report is intended to give useful information to help improve daily laboratory work by making the steps of fly pushing, fluorescent screening, dissection, and documentation/imaging more efficient. It also details various possibilities for properly equipping or stocking a fly lab.
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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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  • STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

    Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS ) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells.
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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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  • Individual Macromolecule Motion in a Crowded Living Cell

    There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data ( FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.
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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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  • What Makes sCMOS Microscope Cameras so Popular?

    sCMOS cameras are more sensitive and are capable of much higher acquisition speed than cameras with other sensor types. Even though CCD cameras are widely used in live cell imaging and time-lapse recordings, researchers are often concerned that their camera does not detect faint signals. In this interview, Dr. Karin Schwab, Product Manager at Leica Microsystems, talks about the characteristics of sCMOS cameras and how researchers benefit from the latest camera sensor technology.
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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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  • How to do a Proper Cell Culture Quick Check

    In order to successfully work with mammalian cell lines, they must be grown under controlled conditions and require their own specific growth medium. In addition, to guarantee consistency their growth must be monitored at regular intervals. This article describes a typical workflow for subculturing an adherent cell line with detailed illustrations of all of the necessary steps.
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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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  • 4Pi-RESOLFT Nanoscopy

    Here we apply the 4Pi scheme to RESOLFT nanoscopy using two-photon absorption for the on-switching of fluorescent proteins. We show that in this combination, the lobes are so low that low-light level, 3D nanoscale imaging of living cells becomes possible. Our method thus offers robust access to densely packed, axially extended cellular regions that have been notoriously difficult to super-resolve. Our approach also entails a fluorescence read-out scheme that translates molecular sensitivity to local off-switching rates into improved signal-to-noise ratio and resolution.
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  • Webinar: Dissecting Protein Dynamics in Living Cells by FRAP

    This webinar presented by Dr Marco Fritzsche, University of Oxford, and Jennifer Horner, PhD, Leica Microsystems, you will learn about how to use Fluorescence Recovery After Photo-bleaching (FRAP) microscopy to study protein dynamics.
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  • Webinar: Using FIT Probes and Super-Resolution Microscopy to Decipher Steps of mRNP Assembly in Developing Oocytes

    In this webinar, presented by Dr. Imre Gaspar of the Developmental Biology Unit at EMBL, you will learn: importance of mRNA localization and function of mRNPs, advantages of using fluorogenic FIT probes to visualize mRNPs in vivo and in fixed specimen, and how super-resolution microscopy can identify factors required for mRNP biogenesis.
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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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  • Probes that FIT RNA

    We have been developing new tools based on fluorogenic forced intercalation (FIT) probes for RNA detection quantification and interference in biological samples. Upon duplex formation with target nucleic acids, the base surrogates TO dye increases its quantum yield and brightness substantially (>10 fold).
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  • Fluorescent Proteins Illuminate Cell Biology

    Green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria and GFP-like fluorescent proteins from other animals have had an important role in the technical innovations that have driven these advances. This poster provides a comprehensive user's guide to fluorescent proteins and sensors , their key properties and the cell biological questions to which they can be applied.
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  • Webinar: Advances in Live Cell Imaging - Skin Cell Motility: Integrins Lead the Way

    In this webinar presented by Leica Microsystems and LabRoots you will learn about efforts to dissect mechanisms that underlie the directed migration of epidermal keratinocytes as they populate wounds or in cancer.
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Useful Links

Communities and Web Sources

www.researchgate.net/
Social network for scientists

www.ibiology.org/
Teaching tools, video lectures on biology and microscopy

bitesizebio.com
Online magazine and community for molecular and cell biology researchers

www.somersault1824.com
Resource for high-end scientific illustrations, images and animations

Search Engines and Data Bases

www.cellimagelibrary.org
Public resource database of images, videos, and animations of cells

harvester.fzk.de/harvester
Bioinformatic meta search engine for genes and proteins

www.gopubmed.com
Search interface for pubmed

en.wikipedia.org/wiki/List_of_academic_databases_and_search_engines
List of academic databases and search engines

scholar.google.com
Beta of Google's search engine for scientific article abstracts

Journals

www.doaj.org/
Directory of open access journals

emboj.embopress.org/
The EMBO Journal

www.lifescied.org
CBE-Life Sciences Education – an ASCB online journal

www.sciencemag.org/
Science

www.nature.com/
Nature

www.cell.com/
Biweekly publication of exceptional research articles

jcs.biologists.org/
Journal of Cell Science

dev.biologists.org/
Development

jeb.biologists.org/
The Journal of Experimental Biology

dmm.biologists.org/
DMM Disease Models & Mechanisms

www.biotechniques.com/
International Journal of Life Science Methods

www.opticsinfobase.org/
Collection of Journals and Proceedings in Optics and Photonics

spie.org/x576.xml
SPIE - peer-reviewed journals on applied research in optics and photonics

onlinelibrary.wiley.com/journal/10.1002/(ISSN)1864-0648
Journal of Biophotonics

www.plosone.org/home.action
International, peer-reviewed, open-access, online publication

rspb.royalsocietypublishing.org/
Proceedings B - the Royal Society's biological research journal

www.microscopy-analysis.com/
International Journal for microscopists

Organizations

www.microscopy.org/
Microscopy Society of America

www.eurmicsoc.org/
European Microscopy Society

www.rms.org.uk/
Royal Microscopical Society

www.ascb.org/
ASCB American Society of Cell Biology

www.biologists.com/cob_activities.html
the company of biologists

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