Andrew Beernink asked: >If you're running X mW of MLUV (light regulated), will a shift in power >among the UV wavelengths change the ratio of Indo (high:low), or >proportionally raise/drop the intensity of both Indo wavelengths I'm >collecting (405 bp & 485 bp)? In other words: in light regulation mode, >based on changes in current, one might get an increase in wavelength A and a >decrease in wavelength B. The question posed is, how will this change >fluorescence? There are substantial differences between the excitation efficiencies of indo-1 with and without bound calcium at 351 and 363 nm (the ratio of excitation at 351 to excitation at 363 changes more for the calcium-saturated than for the calcium-free compound; see the Molecular Probes handbook), so changes in the ratio of these two lines while multiline UV power output remained constant would be expected to cause changes in the 405/485 emission ratio in the absence of changes in intracellular calcium. It would be preferable to use single-line UV. -Howard
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