title: Estimating weak ratiometric signals in imaging data. I. Dual-channel data.
abstract: 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.
authors.affiliation: Center for Applied Mathematics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Josef
authors.initials: J
authors.lastname: Broder
authors.forename: Anirban
authors.initials: A
authors.lastname: Majumder
authors.forename: Erika
authors.initials: E
authors.lastname: Porter
authors.forename: Ganesh
authors.initials: G
authors.lastname: Srinivasamoorthy
authors.forename: Charles
authors.initials: C
authors.lastname: Keith
authors.forename: James
authors.initials: J
authors.lastname: Lauderdale
authors.forename: Andrew
authors.initials: A
authors.lastname: Sornborger
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D015233:Models, Statistical; D016716:PC12 Cells / Q000378:metabolism; D051381:Rats; D012815:Signal Processing, Computer-Assisted; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D002118:Calcium