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Fluorescence Microscopy

Fluorescence microscopy has become the most important physical phenomenon in modern biology and medicine. After a slow start, the possibility of specific fluorescence staining boosted this application in the early 30's of the last century. Around 1950 a second stage was ignited with the invention of immunofluorescent staining. Functional imaging, most prominently by Ca++ probes, delivered the method from the restraint to stick to morphological and structural imaging. The possibility to create living material with targeted expressible fluorescent proteins revolutionized fluorescence microscopy in an unexpected and extensive third stage. Currently, new methods that use specific fluorescent protein-features, are popping up weekly. And last but not least: all modes of super-resolution imaging are based on fluorescence.

  • Acute Transcriptional Up-regulation Specific to Osteoblasts/Osteoclasts in Medaka Fish Immediately after Exposure to Microgravity

    Bone loss is a serious problem in spaceflight; however, the initial action of microgravity has not been identified. To examine this action, we performed live-imaging of animals during a space mission followed by transcriptome analysis using medaka transgenic lines expressing osteoblast and osteoclastspecific promoter-driven GFP and DsRed. In live-imaging for osteoblasts, the intensity of osterix- or osteocalcin-DsRed fluorescence in pharyngeal bones was significantly enhanced 1 day after launch; and this enhancement continued for 8 or 5 days.
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  • The Fundamentals and History of Fluorescence and Quantum Dots

    At some point in your research and science career, you will no doubt come across fluorescence microscopy. This ubiquitous technique has transformed the way in which microscopists can image, tag and trace anything from whole organisms to single proteins and beyond. In this article, we will examine what is meant by "fluorescence", the history and basic physics behind its definition, the discovery and application of Green Fluorescent Protein (GFP) and a look at the rapidly expanding field of fluorescent probes including Quantum Dots.
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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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  • What is Photomanipulation?

    The term photomanipulation describes a wide range of techniques that enable the microscopist the transition from passive observer to instigator of events by offering a way of interacting with their sample via targeted illumination. Typically researchers are trying to observe specific processes of interest in order to understand the underlying biological process. Microscopists are often forced to hunt through large populations of cells or acquire hours of time laps footage before they’re able to observe events of interest and in many cases it’s simply not possible to observe certain processes using conventional microscopy techniques alone. Photomanipulation tools enable the microscopist to initiate biological events, precisely adjusting sample labeling, biological activity, local chemical environments and in some instances physically destroy parts of their specimen.
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  • Photoactivatable, photoconvertible, and photoswitchable Fluorescent Proteins

    Fluorescent proteins (FPs) such as GFP, YFP or DsRed are powerful tools to visualize cellular components in living cells. Nevertheless, there are circumstances when classical FPs reach their limits. Watching dedicated, spatially limited protein populations of a certain protein of interest is impossible with common FPs, since they are expressed throughout the entire cell. At this point photoactivatable, photoconvertible and photoswitchable fluorescent proteins enter the stage. The members of this fluorescence toolkit can be activated from a non-fluorescent state, they can change their emission spectrum, or they are even able to be reversibly switched "on and off". With the help of these “optical highlighters”, researchers can track a distinct protein population over time by activating respectively converting their fluorescence with a spatially defined light beam of a given wavelength.
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  • Work Efficiently in Developmental Biology and Medical Research with Stereo Microscopy: Rodent and Small Animal Surgery

    This report provides information which can help improve the routine work of scientists and technicians performing studies involving surgery on small animals and rodents, i.e. mice, rats, hamsters etc., for developmental biology or medical research. The aim is to help make the work steps efficient and cost-effective, where the employment of microscopes is necessary. It also gives useful hints and details on the various microscopes which can be used in a developmental biology or medical research laboratory where small animal or rodent surgery is exploited.
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  • Milestones in Incident Light Fluorescence Microscopy

    Since the middle of the last century, fluorescence microscopy developed into a bio scientific tool with one of the biggest impacts on our understanding of life. Watching cells and proteins with the help of fluorescence molecules is a standard method in nearly every life science discipline today. This broad application range goes back to the technical work of some researchers who wanted to improve and simplify fluorescence microscopic labor. One person who was involved in that development was the Dutch medic Johann Sebastiaan Ploem.
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  • Five Big Mysteries about CRISPR’s Origins

    Francisco Mojica was not the first to see CRISPR, but he was probably the first to be smitten by it. He remembers the day in 1992 when he got his first glimpse of the microbial immune system that would launch a biotechnology revolution. He was reviewing genome-sequence data from the salt-loving microbe Haloferax mediterranei and noticed 14 unusual DNA sequences, each 30 bases long.
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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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  • Chronic Stress in Mice Remodels Lymph Vasculature to Promote Tumour Cell Dissemination

    Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer.
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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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  • 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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  • Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy as a Straightforward Tool to Study Host-Pathogen Interactions

    Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell.
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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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  • 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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  • Gene Editing with CRISPR/Cas9 - Breakthrough in Genome Engineering

    The CRISPR/Cas9 system is one of several different bacterial systems for defense against viral attacks. It consists of two main components. One is a small piece of RNA which binds to the viral target sequence via Watson-Crick base pairing. It serves as a marker for the foreign nucleic acid. The second component is the Cas9 protein. It binds to the marked sequence and cuts it due to its nuclease activity. Because the base pairing RNA can be synthesized easily and then used to determine a target region, researchers have utilized this system in the laboratory for genome editing.
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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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  • Measuring the 3D STED-PSF with a new Type of Fluorescent Beads

    A new type of fluorescent bead is presented by GATTAquant. These beads, called GATTA-Beads, are characterized by a small diameter (23 nm), high intensity and size uniformity. In combination with state-of the-art STED microscopes such as the Leica TCS SP8 STED 3X and high-end image restoration methods available in the Huygens Software, it is shown that these new beads can be used for accurate STED PSF characterization in 3D. Furthermore, it is shown that the measured 3D STED-PSF can be used to improve image restoration quality in combination with STED deconvolution methods available in the Huygens Software.
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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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  • Video Talk by Roger Tsien: Fluorescent Protein Indicators

    In this talk, Roger Tsien discusses how fluorescent proteins have been turned into indicators for a wide variety of biological molecules, including pH, ions, redox potential, and signaling molecules like phosphoinositides. The talk also covers reporters used to measure the activity of enzymes like kinases, phosphatases, and proteases. It covers both single proteins whose intensity or wavelength change, as well as reporters using resonance energy transfer (FRET).
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  • Video Talk: CRISPR-Cas – From a Bacterial Adaptive Immune System to a Genome Engineering Tool

    The CRISPR-Cas system was originally discovered as an adaptive immune system of bacteria and archaea to protect against viral attack. During this talk, given at Leica Microsystems in Wetzlar, Dr. Lennart Randau, MPI Marburg, presents this simple and fascinating system in detail. Furthermore, he introduces the adaption of the CRISPR-Cas system into a potent molecular biology tool, which is used heavily for genome editing. In addition to its influence on molecular biology, meanwhile the Cas9 nuclease also stimulates microscopy techniques e.g. to fluorescently label genomic loci in living cells.
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  • The Oncogenic Triangle of HMGA2, LIN28B and IGF2BP1 Antagonizes Tumor-Suppressive Actions of the let-7 Family

    The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3′-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3′ UTR shortening.
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  • Time Gating of Chloroplast Autofluorescence Allows Clearer Fluorescence Imaging In Planta

    Chloroplast, an organelle facilitating photosynthesis, exhibits strong autofluorescence, which is an undesired background signal that restricts imaging experiments with exogenous fluorophore in plants. In this study, the autofluorescence was characterized in planta under confocal laser microscopy, and it was found that the time-gated imaging technique completely eliminates the autofluorescence. As a demonstration of the technique, a clearer signal of fluorescent protein-tagged phototropin, a blue-light photoreceptor localized at the chloroplast periphery, was visualized in planta.
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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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  • Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

    Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.
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  • Mammary gland development

    Investigating the Influence of Hormone Signaling Pathways on Mammary Gland Development and the Onset of Breast Cancer

    Over their entire lifetime, 1 out of 8 women can suffer from breast cancer. The risk of developing breast cancer (mammary carcinogenesis) increases with a woman’s age and is related to her reproductive history. The chance of mammary carcinogenesis is less for women who give birth to a child before the age of 30. However, it is now known that the risk of breast cancer also can depend on the number of times a woman experiences elevated blood serum progesterone levels, in relation to the menstrual cycle, before her first pregnancy.
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  • Video Talk by Roger Tsien: Fluorescent Proteins

    Live cell imaging has been revolutionized by the discovery of the green fluorescent protein (GFP). This lecture covers the history of GFP, how it folds and becomes fluorescent, how it has been mutated to produce additional colors (blue, cyan, yellow), and the discovery of red fluorescent proteins from corals. It also covers novel photoswitchable and photoactivatible fluorescent proteins, whose color can be changed by light, and new infrared fluorescent proteins.
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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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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.comOnline magazine and community for molecular and cell biology researchers

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

Search Engines and Data Bases

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

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

www.gopubmed.comSearch interface for pubmed

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

scholar.google.comBeta 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.orgCBE-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.xmlSPIE - peer-reviewed journals on applied research in optics and photonics

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

www.plosone.org/home.actionInternational, 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 / Institutes

www.microscopy.org/Microscopy Society of America

www.eurmicsoc.org/European Microscopy Society

www.rms.org.uk/Royal Microscopical Society

www.doitpoms.ac.ukTutorials on microscopy for materials science; University of Cambridge, Department of Materials Science and Metallurgy

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

www.biologists.com/cob_activities.htmlthe company of biologists

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