Spectral characterization of the effect of viscosity on Fura-2 fluorescence: excitation wavelength optimization abolishes the viscosity artifact.
A systematic study of the spectral characteristics of the viscosity artifact in Fura-2 based [Ca2+] measurements reveals that, by selecting excitation wavelengths approximately 10 nm longer than those routinely employed and modestly reducing excitation bandpasses, the magnitude of the artifact can be reduced to experimentally undetectable levels without greatly impairing [Ca2+] measurements. The feasibility of this approach was confirmed on a ratio imaging microscope; the magnitude of the artifact observed in dextran-conjugated Fura-2 solutions prepared in water or in 50% sucrose was not statistically significant using an excitation wavelength pair of 361/389 nm, whereas at 350/380 nm [Ca2+] was underestimated by 34% in the higher viscosity solution. Thus, provided potential pitfalls are taken into account, a simple change in imaging protocol can avoid the viscosity artifact without recourse to correction factors. This approach may be employed either routinely, or else merely to test whether apparent [Ca2+]i differences observed at more conventional wavelengths arise from the viscosity artifact.
Busa
W B
WB
Department of Biology, Johns Hopkins University, Baltimore, Maryland.
D016477:Artifacts; D002118:Calcium / Q000032:analysis*; D016257:Fura-2 / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014783:Viscosity
D002118:Calcium; D016257:Fura-2