title: Reliable measurement ofE. colisingle cell fluorescence distribution using a standard microscope set-up.
abstract: BACKGROUND
  Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories.
  
  
  METHODS
  A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry.
  
  
  RESULTS
  The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described.
  
  
  CONCLUSIONS
  The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).
authors.affiliation: Department of Electrical, Laboratory of Cellular and Molecular Engineering "S.Cavalcanti", Electronic and Information Engineering "G.Marconi" (DEI), University of Bologna, Cesena, Italy.
authors.forename: Marilisa
authors.identifier: 0000-0002-3731-7760
authors.initials: M
authors.lastname: Cortesi
authors.affiliation: Department of Electrical, Laboratory of Cellular and Molecular Engineering "S.Cavalcanti", Electronic and Information Engineering "G.Marconi" (DEI), University of Bologna, Cesena, Italy.|Present address: SynthSys - Center for Synthetic and Systems Biology and School of Biological Sciences, University of Edinburgh, Edinburgh, UK.
authors.forename: Lucia
authors.initials: L
authors.lastname: Bandiera
authors.affiliation: Department of Electrical, Laboratory of Cellular and Molecular Engineering "S.Cavalcanti", Electronic and Information Engineering "G.Marconi" (DEI), University of Bologna, Cesena, Italy.|Present address: Division of Respiratory Medicine, School of Medicine, University of Nottingham, Nottingham, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Pasini
authors.affiliation: Advanced Research Center on Electronic Systems for Information and Communication Technologies "E. De Castro" (ARCES), University of Bologna, Bologna, Italy.|Department of Computer Science and Engineering (DISI), University of Bologna, Bologna, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bevilacqua
authors.affiliation: Advanced Research Center on Electronic Systems for Information and Communication Technologies "E. De Castro" (ARCES), University of Bologna, Bologna, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Gherardi
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Furini
authors.affiliation: Department of Electrical, Laboratory of Cellular and Molecular Engineering "S.Cavalcanti", Electronic and Information Engineering "G.Marconi" (DEI), University of Bologna, Cesena, Italy.|Advanced Research Center on Electronic Systems for Information and Communication Technologies "E. De Castro" (ARCES), University of Bologna, Bologna, Italy.|BioEngLab, Health Science and Technology, Interdepartmental Center for Industrial Research (HST-CIRI), University of Bologna, Ozzano Emilia, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Giordano
keywords: Fluorescence microscopy; Phenotypic noise; Single-cell fluorescence; Synthetic biology