Posted by
Mark Cannell-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-measurements-for-multiphoton-microscopy-tp7585091p7585101.html
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Hi Chris
Any idea what is the cause of the difference between the measurements you cite and the conclusions of Szmacinski et al (Photochem Photobiol. 1993 Sep;58(3):341-5): "The emission spectra and lifetime of Indo-1 appear to be identical for one-photon and two-photon excitation at 351 and 702 mn, respectively, suggesting that the relative one- and two-photon cross sections are similar for the calcium-free and calcium-bound forms of Indo-1. Also, the two-photon cross section of Indo-1 is relatively high, about 4 x 10(-49) cm4 s/photon molecule at 690 nm for both the calcium-free and calcium-bound forms. “?
Cheers Mark
.
Calcium-dependent fluorescence lifetimes of Indo-1 for one- and two-photon excitation of fluorescence.
Szmacinski H1, Gryczynski I, Lakowicz JR.
On 27/04/2016, at 11:25 am, Christian Wilms <
[hidden email]> wrote:
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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
Mark B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology & Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK
[hidden email]