Ratiometric fluorescent indicators are becoming increasingly prevalent in many areas of biology. They are used for making quantitative measurements of intracellular free calcium both in vitro and in vivo, as well as measuring membrane potentials, pH, and other important physiological variables of interest to researchers in many subfields. Often, functional changes in the fluorescent yield of ratiometric indicators are small, and the signal-to-noise ratio (SNR) is of order unity or less. In particular, variability in the denominator of the ratio can lead to very poor ratio estimates. We present a statistical optimization method for objectively detecting and estimating ratiometric signals in dual-wavelength measurements of fluorescent, ratiometric indicators that improves on standard methods. With the use of an appropriate statistical model for ratiometric signals and by taking the pixel–pixel covariance of an imaging dataset into account, we are able to extract user-independent spatiotemporal information that retains high resolution in both space and time.
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