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Quantitative Fluorescence

The phenomenon of fluorescence has revolutionized research in biology and medicine. More than 100 years ago, Paul Ehrlich began to develop specific chemical staining methods and the latest development of light-controlled proteins (optogenetics) will surely not mark the end of this progression. A historically continuous trend is the quest for measuring and quantifying data, with or without generation of images as data source. Many techniques have been introduced that are based on fluorescence, but not necessarily require image formation. The laboratory jargon refers to this methods as “F-techniques”: FLIM, FCS , FCCS, FRET, FRAP… to mention just the most important. Read the articles in this topic, to find out more details on these modern methods.

  • 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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  • FRAP with TCS SP8 Resonant Scanner

    Fast FRAP experiments need a sufficient number of measurement points for meaningful interpretation and fitting analysis. To study very fast translocational processes, the use of a resonant scanner (RS) is preferred. The advantage in using FRAP with the RS is that statistics are much better in experiments that require fast acquisition: If the half time of recovery is about 0.5 sec you may have only about 3 to 4 data points using the conventional scanner, whereas with the resonant scanner you can get about 20 data points.
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  • Interview with Dr. Gertrude Bunt and Prof. Fred S. Wouters on the FOM 2015

    Only a few days to go before the start of Focus on Microscopy 2015 in Göttingen, Germany. This year’s FOM is being organized by Dr. Gertrude Bunt and Prof. Dr. Fred S. Wouters from the University Medical Center, Göttingen, in cooperation with Prof. Dr. G.J. (Fred) Brakenhoff, University of Amsterdam, The Netherlands.
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  • Integrative Open-Source Software for Image Analysis in Biology

    Imaging techniques are indispensable in many fields of life sciences today. With state-of-the-art optics and metrology, they provide hundreds of gigabytes of still images and videos. Correspondingly, there is a growing need for complex software solutions to ensure that the amounts of generated data can be automatically managed, processed and analyzed – and shared online with a large group of users. The combination of individual open-source software projects is proving especially useful for solving such complex image analysis problems.
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  • The New Repository on the Block

    The need for data validation and accessibility has never been greater than it is today. We are inundated with information from a multitude of resources, but how can we easily evaluate the accuracy of that data? In the past, the peer review process provided this and was often run by publishers.
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  • FLIM-FRET in Solutions

    FRET efficiency can be measured based on fluorescence lifetime microscopy (FLIM). FLIM-FRET allows analysis of molecular interactions both in vitro and in vivo. This article describes the use of FLIM in the time domain (TCSPC) to measure FRET in vitro in a biochemical assay using a Cerulean-Citrine construct.
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  • Choose Your Excitation Wavelength

    Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including a pulsed laser source, a photon counting card, and a fast detector.
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  • From Molecules to Tissues

    Cancer research using confocal and multiphoton microscopy. Sequencing of the human genome stimulated a radical change in the approach to biomedical research. The comprehension of the mechanisms regulating life gained a scale-up in throughput to speed up the retrieval of data for a global vision of a system of incomparable complexity.
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  • 4D Photoactivation of pa-GFP in Living Cells Using Two-Photon Excitation Laser Scanning Microscopy

    We report about two-photon activation of a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (pa-GFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm. Two-photon photoactivation produces an effective real three-dimensional (3D) localization of the molecular switching of pa-GFP in the bright state.
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Search Engines and Data Bases

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Public resource database of images, videos, and animations of cells

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The EMBO Journal

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DMM Disease Models & Mechanisms

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International Journal of Life Science Methods

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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

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Proceedings B - the Royal Society's biological research journal

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