2D luminescence imaging of pH in vivo

Luminescence imaging of biological parameters is an emerging field in biomedical sciences. Tools to study 2D pH distribution are needed to gain new insights into complex disease processes, such as wound healing and tumor metabolism. In recent years, luminescence-based methods for pH measurement have been developed. However, for in vivo applications, especially for studies on humans, biocompatibility and reliability under varying conditions have to be ensured. Here, we present a referenced luminescent sensor for 2D high-resolution imaging of pH in vivo. The ratiometric sensing scheme is based on time-domain luminescence imaging of FITC and ruthenium(II)tris-(4,7-diphenyl-1,10-phenanthroline). To create a biocompatible 2D sensor, these dyes were bound to or incorporated into microparticles (aminocellulose and polyacrylonitrile), and particles were immobilized in polyurethane hydrogel on transparent foils. We show sensor precision and validity by conducting in vitro and in vivo experiments, and we show the versatility in imaging pH during physiological and chronic cutaneous wound healing in humans. Implementation of this technique may open vistas in wound healing, tumor biology, and other biomedical fields.

[1]  V. Bindokas,et al.  Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages* , 2009, The Journal of Biological Chemistry.

[2]  Pernille R. Jensen,et al.  Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate , 2008, Nature.

[3]  M. Landthaler,et al.  Wound healing in the 21st century. , 2010, Journal of the American Academy of Dermatology.

[4]  R S Sobel,et al.  Fluorescein angiography complication survey. , 1986, Ophthalmology.

[5]  D. Greenhalgh,et al.  Cutaneous Wound Healing , 2007, Journal of burn care & research : official publication of the American Burn Association.

[6]  Timothy J. Mitchison,et al.  A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish , 2009, Nature.

[7]  A. Miyawaki,et al.  Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Matthias I. J. Stich,et al.  Multiple fluorescent chemical sensing and imaging. , 2010, Chemical Society reviews.

[9]  P. Okunieff,et al.  Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. , 1989, Cancer research.

[10]  Ian Constable,et al.  Fluorescein angiography and adverse drug reactions revisited: the Lions Eye experience , 2006, Clinical & experimental ophthalmology.

[11]  Enrico Gratton,et al.  NHE1 Regulates the Stratum Corneum Permeability Barrier Homeostasis , 2002, The Journal of Biological Chemistry.

[12]  K. Davey,et al.  Modelling the combined effect of temperature and pH on the rate coefficient for bacterial growth. , 1994, International journal of food microbiology.

[13]  I Klimant,et al.  Fluorescent imaging of pH with optical sensors using time domain dual lifetime referencing. , 2001, Analytical chemistry.

[14]  W. R. Taylor,et al.  In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles. , 2007, Nature materials.

[15]  E. Olson.  Influence of pH on bacterial gene expression , 1993, Molecular microbiology.

[16]  Avner Friedman,et al.  A mathematical model of ischemic cutaneous wounds , 2009, Proceedings of the National Academy of Sciences.

[17]  M. Ohkura,et al.  A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein , 2001, Nature Biotechnology.

[18]  A. Vahlquist,et al.  In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. , 1994, Acta dermato-venereologica.

[19]  G. Winter,et al.  Formation of the Scab and the Rate of Epithelization of Superficial Wounds in the Skin of the Young Domestic Pig , 1962, Nature.

[20]  T. Dahl,et al.  Readily available fluorescein isothiocyanate-conjugated antibodies can be easily converted into targeted phototoxic agents for antibacterial, antiviral, and anticancer therapy. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Altman,et al.  STATISTICAL METHODS FOR ASSESSING AGREEMENT BETWEEN TWO METHODS OF CLINICAL MEASUREMENT , 1986, The Lancet.

[22]  Christian Krause,et al.  Composite Luminescent Material for Dual Sensing of Oxygen and Temperature , 2006 .

[23]  Natarajan Raghunand,et al.  In vivo imaging of extracellular pH using 1H MRSI , 1999, Magnetic resonance in medicine.

[24]  Jonathan Hadgraft,et al.  Epidermal barrier dysfunction in atopic dermatitis. , 2009, The Journal of investigative dermatology.

[25]  A. Beckett,et al.  AKUFO AND IBARAPA. , 1965, Lancet.

[26]  L Reinisch,et al.  Intracellular pH measurement using single excitation-dual emission fluorescence ratios. , 1990, The American journal of physiology.

[27]  Timothy A. Dall,et al.  The burden of skin diseases: 2004 a joint project of the American Academy of Dermatology Association and the Society for Investigative Dermatology. , 2006, Journal of the American Academy of Dermatology.

[28]  Michael Landthaler,et al.  The impact of the pH value on skin integrity and cutaneous wound healing , 2010, Journal of the European Academy of Dermatology and Venereology : JEADV.

[29]  E. Arriaga,et al.  Individual acidic organelle pH measurements by capillary electrophoresis. , 2006, Analytical chemistry.

[30]  M. Dewhirst,et al.  A dual-emissive-materials design concept enables tumour hypoxia imaging. , 2009, Nature materials.

[31]  T. K. Hunt,et al.  Respiratory gas tensions and pH in healing wounds. , 1967, American journal of surgery.

[32]  J M Bland,et al.  Statistical methods for assessing agreement between two methods of clinical measurement , 1986 .

[33]  R. Gillies,et al.  31P-MRS measurements of extracellular pH of tumors using 3-aminopropylphosphonate. , 1994, The American journal of physiology.

[34]  M. Schnitzer,et al.  In vivo fluorescence imaging with high-resolution microlenses , 2009, Nature Methods.

[35]  Avner Friedman,et al.  Wound angiogenesis as a function of tissue oxygen tension: A mathematical model , 2008, Proceedings of the National Academy of Sciences.

[36]  H. Sorg,et al.  Wound Repair and Regeneration , 2012, European Surgical Research.

[37]  R. Niesner,et al.  3D-Resolved Investigation of the pH Gradient in Artificial Skin Constructs by Means of Fluorescence Lifetime Imaging , 2005, Pharmaceutical Research.

[38]  Carlsson,et al.  Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity‐modulated multiple‐wavelength scanning (IMS) technique , 1998, Journal of microscopy.

[39]  Tumour pH , 1992, The Lancet.

[40]  I Klimant,et al.  Inert phosphorescent nanospheres as markers for optical assays. , 2001, Bioconjugate chemistry.