Second harmonic imaging of membrane potential of neurons with retinal

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Abstract
We present a method to optically measure and image the membrane potential of neurons, using the nonlinear optical phenomenon of second harmonic generation (SHG) with a photopigment retinal as the chromophore [second harmonic retinal imaging of membrane potential (SHRIMP)]. We show that all-trans retinal, when adsorbed to the plasma membrane of living cells, can report on the local electric field via its change in SHG. Using a scanning mode-locked Ti-sapphire laser, we collect simultaneous two-photon excited fluorescence (TPEF) and SHG images of retinal-stained kidney cells and cultured pyramidal neurons. Patch clamp experiments on neurons stained with retinal show an increase of 25% in SHG intensity per 100-mV depolarization. Our data are the first demonstration of optical measurements of membrane potential of mammalian neurons with SHG. SHRIMP could have wide applicability in neuroscience and, by modifying rhodopsin, could in principle be subject for developing genetically engineered voltage sensors. © 2004 Society of Photo-Optical Instrumentation Engineers.