BME 210 Lecture 7 Imaging of Intracellular Calcuim

BME 210 Lecture 7 Imaging of Intracellular Calcuim - 7...

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7. Imaging of Intracellular Calcium
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Roles of Ca ions Ca 2+ ions - “second messengers” (intracellular regulators) participating in numerous cell processes including: cell development intracellular signaling muscle contraction synaptic transmission; memory ion channel gating secretion, etc.
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Measurement of intracellular [Ca 2+ ] Currently, dynamic measurements of intracellular [Ca 2+ ] are possible only using fluorescent Ca 2+ -sensitive dyes Types of Ca 2+ -sensitive dyes: 1. Fluorescent organic dyes 2. Photoproteins : - Aequorin - GFP-based genetically engineered FPs: “Cameleons” and “Pericams”
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Organic Ca-sensitive dyes Dye Ex (nm) Em (nm) K d (nM) Indo-1 350 400/480 230 Fura-2 340/380 510 145 Fluo-4 495 515 345 Rhod-2 530 580 570 Ca Green 506 531 190 Ca Crimson 590 615 185 • Total of ≈ 50 dyes (Invitrogen – Molecular Probes) “Ratiometric” Non- ratiometric
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Membrane permeability Original Ca dyes are polar and membrane impermeable Membrane-permeable derivatives are made by attaching ester groups, which make molecules neutral, e.g.,: Fluo-4 Fluo-4 AM (AcetoxyMethyl ester derivative) The Fluo-4 AM dye is membrane permeable but doesn’t bind Ca 2+ The ester groups are removed inside cells by esterases, making the dye functional Fura-2 AM Fura-2
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Ca waves in oocytes • Starfish oocyte stained with Ca-Green dye • Confocal images taken at 5-s interval • [Ca] wave was initiated by fertilization
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Ca waves and transients in the heart • Rat heart • Fluo-3 AM staining • Real-time confocal imaging Ca transient
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Ca-sensitive photoproteins Aequorin Aequorin luminescent protein isolated from jellyfish Aequorea victoria • Binding of Ca 2+ change of protein conformation emission of blue light (469 nm) • Discovered in 1962; used in first measurements of intracellular Ca in 1967
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Fluorescence Resonance Energy Transfer (FRET) 2 fluorescent dyes with overlapping spectra: – dye 1 (donor) is excited by incident light
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BME 210 Lecture 7 Imaging of Intracellular Calcuim - 7...

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