Re: Ratiometric measurements for multiphoton microscopy

Posted by Christian Wilms on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-measurements-for-multiphoton-microscopy-tp7585091p7585100.html

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"When we used info-1 with 690 nm 2P excitation, I thought that the ratio image was quite reasonable (see Jones, K.T., Soeller, C., Cannell, M.B., 1998. The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse. Development 125, 4627-4635)."

I admit that I never tried it in cells, only cuvettes. But I could reproduce the more than an order of magnitude lower excitation efficiency than e.g. Fura-2 (http://www.drbio.cornell.edu/cross_sections.html) and decided that the required excitation power was not ideal for my experiments on small neuronal compartments. When imaging larger structures at low zoom, that should be much less of a problem.

"Also, using a second Ca insensitive dye to construct a "ratio" measurement is unwise -the point of the second wavelength is to control for bleaching and dye distribution etc."

I am also not at all a fan of the approach. It comes with numerous problems, starting with different diffusion rates of the two dyes over different bleaching behaviour, to differential scattering of the two emission wavelengths, leading to a depth-dependent gradient in the ratio, that would need to be corrected for. Having said all that, it has become pretty much standard in neuroscience to report 'green over red' ratios. See publications by the Sabatini for examples.

Best, Chris