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• W2021001524 abstract "In recent years, quantification of absolute protein numbers in cellular structures using fluorescence microscopy has become a reality. Two popular methods are available to a broad range of researchers with minimal equipment and analysis requirements: stepwise photobleaching to count discrete changes in intensity from a small number of fluorescent fusion proteins, and comparing the fluorescence intensity of a protein to a known in vivo or in vitro standard. This review summarizes the advantages and disadvantages of each method, and gives recent examples of each that answer important questions in their respective fields. We also highlight new counting methods that could become widely available in the future. In recent years, quantification of absolute protein numbers in cellular structures using fluorescence microscopy has become a reality. Two popular methods are available to a broad range of researchers with minimal equipment and analysis requirements: stepwise photobleaching to count discrete changes in intensity from a small number of fluorescent fusion proteins, and comparing the fluorescence intensity of a protein to a known in vivo or in vitro standard. This review summarizes the advantages and disadvantages of each method, and gives recent examples of each that answer important questions in their respective fields. We also highlight new counting methods that could become widely available in the future. reversible loss of emission intensity from FPs due to transition to a nonemissive triplet state more likely to occur at higher excitation intensities. the best resolution that can be obtained by a light microscope, given by optical emission wavelength (λ) divided by two times the numerical aperture (N.A.) of the objective lens (λ/2N.A.); ∼200 nm at best. a process by which cells or microscopic particles in suspension flow past a detector one at a time and the detector counts the number and records the fluorescence intensity and other parameters. a technique in which fluctuations of fluorescence intensity are measured within a small volume and physical properties (e.g., rate of diffusion, concentration of molecules, interactions) of the fluorescent molecules passing through that volume can be mathematically extracted using autocorrelation functions. the gene for a fluorescent protein, such as GFP, is inserted in frame up- or downstream of the gene for a protein of interest, so that when transcribed and translated, the resulting protein of interest is fused to GFP. energy transfer from a donor fluorophore to an acceptor fluorophore in close proximity (<10 nm and depending on the alignment of the fluorophores with respect to one another) when the donor emission wavelength overlaps the acceptor excitation wavelength. on a Gaussian curve, the width of the curve at a height that is half the maximum height. The FWHM of the point spread function approximates Z axis or axial resolution. the time it takes for a fluorophore, such as GFP, to mature to its fluorescent state via rearrangements and chemical reactions among amino acids. inconstant imprecise output above and below a real signal that disturbs or interferes with detection of the signal, usually referred to as ‘snow’ on a television screen when the broadcast signal is lost. irreversible loss of fluorescence due to exposure to an excitation light source. the apparent blurring of intensity from a point source of light, such as a fluorescent bead or protein, due to diffraction of light by the lens. any technique that breaks the diffraction limit of fluorescence microscopy (∼200 nm) by pinpointing the exact location of point sources and representing the image using those locations rather than the additive point spread functions of all point sources in an imaging field. a protein constructed by fusing a truncated transcription activator-like effector to FokI restriction enzyme. Each TALE repeat binds a single DNA base pair, making them more versatile than zinc fingers. TALENs can be used for high-throughput genome editing. a protein constructed by fusing a zinc finger, which is a DNA binding motif, to a restriction enzyme; usually FokI is used because it has a nonspecific cleavage site. Zinc fingers can be combined to recognize specific sequences of DNA on either side of a desired cleavage site, and FokI dimerizes in the middle and creates a double-stranded break. Existing DNA repair mechanisms can utilize homologous sequences supplied exogenously to insert DNA while repairing the break." @default.
• W2021001524 created "2016-06-24" @default.
• W2021001524 creator A5062935470 @default.
• W2021001524 creator A5066026692 @default.
• W2021001524 date "2012-11-01" @default.
• W2021001524 modified "2023-09-23" @default.
• W2021001524 title "Counting protein molecules using quantitative fluorescence microscopy" @default.
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• W2021001524 doi "https://doi.org/10.1016/j.tibs.2012.08.002" @default.
• W2021001524 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3482307" @default.
• W2021001524 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22948030" @default.
• W2021001524 hasPublicationYear "2012" @default.
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