Image acquisition and analysis. Images were recorded using atwo-photon laser scanning microscope (Bio-Rad MRC-1024MP)with excitation at 740 nm (Tsunami Ti : Sapphire laser, Spectra-Physics) as described before (Aon et al., 2003). Briefly, owing to theoverlap in the cross-sections for two-photon excitation of the threefluorophores of interest (Xu et al., 1996) (TMRE, CM-DCF, andNADH or GSB) this wavelength permitted recording of Dym, ROSproduction, and NAD(P)H or GSH simultaneously. The red emissionof TMRE was collected at 605±25 nm; the green emission ofCM-DCF was recorded at 525±25 nm; and the blue emission ofGSB detected at 480±20 nm. NADH emission was collected as thetotal fluorescence <490 nm. At 3?5 s or 30 s intervals as indicated,5126512 pixel 8-bit greyscale images of the three emission channelswere collected simultaneously and stored.
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