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Dear list members,
I am having an issue with the IR laser (Chameleon Ultra) power when trying to image dendrites in acute brain slices (~50 micrometers deep into tissue) using the LSM 510 NLO.
Namely, to obtain any decent signal with either descanned (open PH) or non-descanned detector, I must use 5-10% laser power = 50-100 mW, which kills the cells with a few shots (I can not even start doing ANY experiment).
I can do it with the Argon laser at 5% laser power (=1.5 mW) or less, depending on binning (PH~300; detector gain ~700), using the same dye at the same concentration (OG Bapta 6F, 200-300nM; or Fluo 5F, 200-300 nM; applied through the patch pipette, filling time - 15 minutes to 1 hour; pipette solution: (mM) 110 K-gluconate, 10 KCl, 10 HEPES, 10 Na2-Phosphocreatine, 2 Mg1.3-ATP, 0.2 Na3-GTP; pH 7.2, 290 mOsm; bath solution - ACSF - 124 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 10 D-glucose).
What may be wrong???
Thank you
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