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Fluorescence Microscopy

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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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  • Mission Impossible Accomplished: Tunable Colors for Non-descanning Detection

    Leica Microsystems’ 4Tune detector, the key component of the SP8 DIVE Deep In Vivo Explorer, provides spectrally tunable image recording with non-descanning detection. An innovative solution for multiparameter multiphoton microscopy. The colorful image on the right shows multiphoton microscopy of an unstained mouse skin section acquired using the 4Tune detector. The green color codes for autofluorescence of muscle tissue. Red shows second harmonic generation of fibers upon illumination with 900 nm. The blue pattern is generated by third harmonic generation at lipid boundaries from illumination at 1230 nm.
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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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  • 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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  • 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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