Measuring changes in ion concentration by ratio fluorescence imaging Nicolas DemaurexDepartment of Cell Physiology and MetabolismUniversity of Geneva, SwitzerlandèmeIII Cycle Romand en sciences biologiquesIntroduction to microimaging techniques Geneva, June 7, 2006Menu• Ion imaging: probes and tools• Ratiometric measurements• Calcium imaging• pH imaging of intracellular organellesIon imaging2+Ca signals coded in• Time (msec to days)• Space (nm to cm)• Frequency • Amplitude Cardiac myocytes, Fluo-4, 20 Hz. 500 msec1Ion-sensitive dyes• The fluorophorecontains a chelatormoiety (EGTA) or titrable groups (BCECF)2+Ca• Ion binding alters the fluorophoreQuantum Yield or Stokes shiftSingle wavelength indicators• Ion binding alters Quantum Yield• Number of emitted photons varies with ion concentration• Spectral response preservedRatiometric indicators• Ion binding altersStokes shift• Energy of converted photons varies with ion concentration• Spectral response altered2Ratiometric imagingAllows to correct for: • Changes in focus, cell thickness, loading• Changes in refractive index, viscosity, pH• PhotobleachingCalcium indicators•N on R atiom etric Fluo-3, Fluo-4, Calcium green, Fura Red• Excitation Ratio Fura-2, Mag-fura-2, BTC• Emission Ratio Indo-1, Mag-indo-1Loading cells with fluorophoresInvasive methods Non-invasive methods• Microinjection • Acetoxymethyl (AM) ester loading• Scrape-loading• Acid ...
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