title: Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy.
abstract: Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.
authors.affiliation: Indiana University School of Medicine; Indianapolis, IN USA ; The Roudebush VA; Indianapolis, IN USA.
authors.forename: Ruben M
authors.initials: RM
authors.lastname: Sandoval
authors.affiliation: Department of Cellular and Structural Biology; University of Texas Health Science Center; San Antonio, TX USA.
authors.forename: Exing
authors.initials: E
authors.lastname: Wang
authors.affiliation: Indiana University School of Medicine; Indianapolis, IN USA ; The Roudebush VA; Indianapolis, IN USA.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Molitoris
keywords: 2-photon; albumin; black level; low intensity; offset; permeability; sensitivity