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Active Illumination Microscopy for Live-Cell Imaging

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Neil Anderson, Semrock Inc., and Kengyeh Chu and Jerome Mertz, Boston University

Photobleaching and phototoxicity are unavoidable in fluorescence microscopy. The phenomenon of photobleaching occurs when, as a result of photo-induced modifications to the molecular structure, a fluorophore permanently loses the ability to fluoresce. Phototoxicity arises from photoinduced generation of singlet oxygen and/or other toxic by-products. Both phenomena are detrimental to fluorescence microscopy applications, especially in the case of live-cell imaging, where real-time effects can impair the cell under study. Therefore it is critical to reduce these consequences to a negligible...Read full article

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    Published: November 2010
    Glossary
    aperture
    An opening or hole through which radiation or matter may pass.
    avalanche photodiode
    A device that utilizes avalanche multiplication of photocurrent by means of hole-electrons created by absorbed photons. When the device's reverse-bias voltage nears breakdown level, the hole-electron pairs collide with ions to create additional hole-electron pairs, thus achieving a signal gain.
    detector
    1. A device designed to convert the energy of incident radiation into another form for the determination of the presence of the radiation. The device may function by electrical, photographic or visual means. 2. A device that provides an electric output that is a useful measure of the radiation that is incident on the device.
    fluorescence
    Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
    fluorescence microscopy
    Fluorescence microscopy is a specialized optical imaging technique used in biology, chemistry, and materials science to visualize and study specimens that exhibit fluorescence. Fluorescence is the phenomenon where a substance absorbs light at one wavelength and emits light at a longer wavelength. In fluorescence microscopy, fluorescent dyes or proteins are used to label specific structures or molecules within a sample. The basic principles of fluorescence microscopy involve illuminating the...
    fluorophore
    A fluorophore is a molecule or a portion of a molecule that has the ability to emit light upon excitation by an external energy source, such as ultraviolet or visible light. The process by which a fluorophore absorbs and then re-emits light is known as fluorescence. Fluorophores are widely used in various scientific and technological applications, including fluorescence microscopy, flow cytometry, medical imaging, and molecular biology. Key features of fluorophores include: Excitation and...
    microscope objective
    The lens located at the object end of a microscope tube. A wide range of objectives is available, including simple achromats, the color-corrected apochromats and the flat-field objectives. Objectives are specified by numerical aperture and magnification (at the standard 160-mm tube length).
    photobleaching
    Photobleaching is a phenomenon in which the fluorescence of a fluorophore (a fluorescent molecule or dye) is permanently reduced or eliminated upon prolonged exposure to light. This process occurs due to the photochemical destruction or alteration of the fluorophore molecules, rendering them non-fluorescent. Key points about photobleaching: Mechanism: Photobleaching is typically a result of chemical reactions induced by the absorbed photons. The excessive light exposure causes the...
    photomultiplier tube
    A photomultiplier tube (PMT) is a highly sensitive vacuum tube that detects and amplifies low levels of light. It is widely used in various applications where high sensitivity, fast response times, and low-light detection capabilities are crucial. Photomultiplier tubes are particularly valuable in scientific research, medical diagnostics, and industrial instrumentation. Key features and principles of PMTs include: Photoelectric effect: The operation of a photomultiplier tube is based on the...
    phototoxicity
    Phototoxicity refers to the harmful effects caused by exposure to light, particularly intense or ultraviolet (UV) light, on living cells or organisms. This phenomenon is often associated with the interaction of light with certain substances, known as photosensitizers, which can lead to cellular damage or death. Phototoxic reactions can occur in various biological systems, including cells, tissues, and organisms. Key points about phototoxicity include: Photosensitizers: Photosensitizers are...
    signal-to-noise ratio
    The ratio of the power in a desired signal to the undesirable noise present in the absence of a signal.
    spatial resolution
    Spatial resolution refers to the level of detail or granularity in an image or a spatial dataset. It is a measure of the smallest discernible or resolvable features in the spatial domain, typically expressed as the distance between two adjacent pixels or data points. In various contexts, spatial resolution can have slightly different meanings: Imaging and remote sensing: In the context of satellite imagery, aerial photography, or other imaging technologies, spatial resolution refers to the...
    active illumination microscopyAIMapertureavalanche photodiodeBiophotonicsBoston Universitydetectorexcitation lightFeaturesfeedback activeFiltersfluorescencefluorescence microscopyfluorophoreGFPhippocampusillumination microscopyimage saturationimagesImagingimaging tool kitJerome MertzKengyeh Chulaser powerlaser scanning fluorescence microscopylaser sourcelinear imagelive-cell imagingmicroscope objectiveMicroscopymouse brainNeil AndersonOpticsphotobleachingphotoinduced modificationsphotomultiplier tubePhototoxicitypixelsPMTpower limitedSemrock Inc.Sensors & Detectorssignal-to-noise ratioSNRspatial resolutionsubpixel timescaleTPEF microscopytwo-photon microscope

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