Annals of Biomedical Engineering

, Volume 31, Issue 9, pp 1084–1096 | Cite as

Oxygen Tension Imaging in the Mouse Retina

  • Ross D. Shonat
  • Amanda C. Kight


A newly developed microscope-based imaging system was used to measure the oxygen tension (PO2) inside the retinal and choroidal vessels of mice and to generate in vivo maps of retinal PO2. These maps were generated from the phosphorescence lifetimes of an injected palladium–porphyrin compound using a frequency-domain measurement. The system was fully calibrated and used to produce retinal PO2 maps at different inspiratory oxygen fractions. PO2 rose accordingly and predictably as inspiratory O2 was stepped from hypoxic to hyperoxic conditions. Important experimental and acquisition parameters necessary for applying phosphorescence lifetime imaging to the mouse eye were investigated, including camera exposure and intensifier gain settings. Because of a need to limit light exposure to the retina, PO2 map quality as measured by the coefficient of determination was investigated as a function of signal-to-noise and accumulated excitation energy deposition. With the development of this technology for use in mice, the potential for investigating the oxygen dynamics in genetically engineered mouse models of retinal disease, including diabetic retinopathy, glaucoma, and age-related macular degeneration, is advanced. © 2003 Biomedical Engineering Society.

PAC2003: 4266Ew, 8763Lk, 8719Dd

Phosphorescence lifetime Phosphorescence quenching Phase-sensitive detection Palladium-porphyrin compound 


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  1. 1.
    Alder, V. A., S. J. Cringle, and I. J. Constable. The retinal oxygen profile in cats. Invest. Ophthalmol. Visual Sci.24:30–36, 1983.Google Scholar
  2. 2.
    Alder, V. A., E.-N. Su, D.-Y. Yu, S. J. Cringle, and P. K. Yu. Diabetic retinopathy: Early functional changes. Clin. Exp. Pharmacol. Physiol.24:785–788, 1997.Google Scholar
  3. 3.
    Alm, A., and A. Bill. The oxygen supply to the retina. I. Effects of changes in intraocular and arterial blood pressures, and in arterial pO2 and pCO2 on the oxygen tension in the vitreous body of the cat. Acta Physiol. Scand.84:261–274, 1972.Google Scholar
  4. 4.
    Amin, R. H., R. N. Frank, A. Kennedy, D. Elliot, J. E. Pulkin, and G. W. Abrams. Vascular endothelial growth factor is present in glial cells of the retina and optic nerve of human subjects with nonproliferative diabetic retinopathy. Invest. Ophthalmol. Visual Sci.38:2729–2741, 1997.Google Scholar
  5. 5.
    Beach, J. M., K. J. Schwenzer, S. Srinivas, D. Kim, and J. S. Tiedeman. Oximetry of retinal vessels by dual-wavelength imaging: Calibration and influence of pigmentation. J. Appl. Physiol.86:748–758, 1999.Google Scholar
  6. 6.
    Berkowitz, B. A., and C. A. Wilson. Quantitative mapping of ocular oxygenation using magnetic resonance imaging. Magn. Reson. Med.33:579–581, 1995.Google Scholar
  7. 7.
    Berkowitz, B. A., C. A. Wilson, and D. L. Hatchell. Oxygen kinetics in the vitreous substitute perfluorotributylamine: A 19F-NMR study. Invest. Ophthalmol. Visual Sci.32:2382–2387, 1991.Google Scholar
  8. 8.
    Bonhoeffer, T., and A. Grinvald. Optical imaging based on intrinsic signals: The methodology. In: Brain Mapping: The Methods, edited by A. W. Toga and J. C. Mazziotta. San Diego: Academic, 1996, pp. 55–97.Google Scholar
  9. 9.
    Buerk, D. G., R. D. Shonat, C. E. Riva, and S. D. Cranstoun. O2 gradients and countercurrent exchange in the vitreous humor near retinal arterioles and venules. Microvasc. Res.45:134–148, 1993.Google Scholar
  10. 10.
    Buxton, R. B. Commentary: The elusive initial dip. Neuroimage13:953–958, 2001.Google Scholar
  11. 11.
    Campochiaro, P. A. Retinal and choroidal neovascularization. J. Cell Physiol.184:301–310, 2000.Google Scholar
  12. 12.
    Crittin, M., H. Schmidt, and C. E. Riva. Hemoglobin oxygen saturation (sO2) in the human ocular fundus measured by reflectance oximetry: Preliminary data in retinal veins. Klin. Monatsbl. Augenheilkd.219:289–291, 2002.Google Scholar
  13. 13.
    Duong, T. Q., S.-C. Ngan, K. Ugurbil, and S.-G. Kim. Functional magnetic resonance imaging of the retina. Invest. Ophthalmol. Visual Sci.43:1176–1181, 2002.Google Scholar
  14. 14.
    Golub, A. S., and R. N. Pittman. Recovery of radial pO2 profiles from phosphorescence quenching measurements in microvessels. Comp. Biochem. Physiol. A132:169–176, 2002.Google Scholar
  15. 15.
    Golub, A. S., A. S. Popel, L. Zheng, and R. N. Pittman. Analysis of phosphorescence decay in heterogeneous systems: Consequences of finite excitation flash duration. Photochem. Photobiol.69:624–632, 1999.Google Scholar
  16. 16.
    Hayreh, S. S. The pathogenesis of optic nerve lesions in glaucoma. Trans. Am. Acad. Ophthalmol. Otolaryngol.81:OP-197–OP-213, 1976.Google Scholar
  17. 17.
    Kim, D.-S., T. Q. Duong, and S.-G. Kim. High-resolution mapping of iso-orientation columns by fMRI. Nature Neuroscience3:164–169, 2000.Google Scholar
  18. 18.
    Lakowicz, J. R., and K. W. Berndt. Lifetime-selective fluorescence imaging using a rf phase-sensitive camera. Rev. Sci. Instrum.62:1727–1734, 1991.Google Scholar
  19. 19.
    Lakowicz, J. R., H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson. Fluorescence lifetime imaging. Anal. Biochem.202:316–330, 1992.Google Scholar
  20. 20.
    Lanni, F., and T. Wilson. Grating image systems for optical sectioning fluorescence microscopy of cells, tissues, and small organisms. In: Imaging Neurons: A Laboratory Manual, edited by R. Yuste, F. Lanni, and A. Konnerth. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2000, pp. 8.1–8.9.Google Scholar
  21. 21.
    Linsenmeier, R. A.Effects of light and darkness on oxygen distribution and consumption in the cat retina. J. Gen. Physiol.88:521–542, 1986.Google Scholar
  22. 22.
    Linsenmeier, R. A., R. D. Braun, M. A. McRipley, L. B. Padnick, J. Ahmed, D. L. Hatchell, D. S. McLeod, and G. A. Lutty. Retinal hypoxia in long-term diabetic cats. Invest. Ophthalmol. Visual Sci.39:1647–1657, 1998.Google Scholar
  23. 23.
    Lo, L.-W., C. J. Koch, and D. F. Wilson. Calibration of oxygen-dependent quenching of the phosphorescence of Pd-meso-tetra (4–carboxyphenyl) porphine: A phosphor with general application for measuring oxygen concentration in biological systems. Anal. Biochem.236:153–160, 1996.Google Scholar
  24. 24.
    Lutty, G. A., D. S. McLeod, C. Merges, A. Diggs, and J. Plouet. Localization of VEGF in human retina and choroid. Arch. Ophthalmol. (Chicago)114:971–977, 1996.Google Scholar
  25. 25.
    Patz, A.Current concepts of the effect of oxygen on the developing retina. Curr. Eye Res.3:159–163, 1984.Google Scholar
  26. 26.
    Riva, C. E., S. D. Cranstoun, R. M. Mann, and G. E. Barnes. Local choroidal blood flow in the cat by laser Dopper flowmetry. Invest. Ophthalmol. Visual Sci.35:608–618, 1994.Google Scholar
  27. 27.
    Riva, C. E., G. T. Feke, B. Eberli, and V. Benary. Bidirectional LDV system for absolute measurement of blood speed in retinal vessels. Appl. Opt.18:2302–2306, 1979.Google Scholar
  28. 28.
    Riva, C. E., C. J. Pournaras, and M. Tsacopoulos. Regulation of local oxygen tension and blood flow in the inner retina during hyperoxia. J. Appl. Physiol.61:592–598, 1986.Google Scholar
  29. 29.
    Rumsey, W. L., M. Pawlowski, N. Lejavardi, and D. F. Wilson. Oxygen pressure distribution in the heart in vivo and evaluation of the ischemic “border zone.” Am. J. Physiol.266:H1676–H1680, 1994.Google Scholar
  30. 30.
    Sakaue, H., Y. Tsukahara, A. Negi, N. Ogino, and Y. Honda. Measurement of vitreous oxygen tension in human eyes. Jpn. J. Ophthalmol.33:199–203, 1989.Google Scholar
  31. 31.
    Schwartz, B., J. C. Rieser, and S. L. Fishbein. Fluorescein angiographic defects of the optic disc in glaucoma. Arch. Ophthalmol. (Chicago)95:1961–1974, 1977.Google Scholar
  32. 32.
    Shonat, R. D., and P. C. Johnson. Oxygen tension gradients and heterogeneity in the venous microcirculation: A phosphorescence quenching study. Am. J. Physiol.272:H2233–H2240, 1997.Google Scholar
  33. 33.
    Shonat, R. D., E. S. Wachman, W.-H. Niu, A. P. Koretsky, and D. L. Farkas. Near-simultaneous hemoglobin saturation and oxygen tension maps in mouse brain using an AOTF microscope. Biophys. J.73:1223–1231, 1997.Google Scholar
  34. 34.
    Shonat, R. D., D. F. Wilson, C. E. Riva, and S. D. Cranstoun. Effect of acute increases in intraocular pressure on intravascular optic nerve head oxygen tension in cats. Invest. Ophthalmol. Visual Sci.33:3174–3180, 1992.Google Scholar
  35. 35.
    Shonat, R. D., D. F. Wilson, C. E. Riva, and M. Pawlowski. Oxygen distribution in the retinal and choroidal vessels of the cat as measured by a new phosphorescence imaging method. Appl. Opt.31:3711–3718, 1992.Google Scholar
  36. 36.
    Silva, A. C., S.-P. Lee, C. Iadecola, and S.-G. Kim. Early temporal characteristics of cerebral blood flow and deoxyhemoglobin changes during somatosensory stimulation. J. Cereb. Blood Flow Metab.20:201–206, 2000.Google Scholar
  37. 37.
    Sinaasappel, M., and C. Ince. Calibration of Pd–porphyrin phosphorescence for oxygen concentration measurements. J. Appl. Physiol.81:2297–2303, 1996.Google Scholar
  38. 38.
    Smith, L. E. H., E. Wesolowski, A. McLellan, S. K. Kostyk, R. D'Amato, R. Sullivan, and P. A. D'Amore. Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Visual Sci.35:101–111, 1994.Google Scholar
  39. 39.
    Stefánsson, E.Oxygen and diabetic eye disease. Graefe's Arch. Clin. Exp. Ophthalmol.228:120–123, 1990.Google Scholar
  40. 40.
    Stefánsson, E., R. Machemer, E. de Juan, Jr., B. W. McCuen, and J. Peterson. Retinal oxygenation and laser treatment in patients with diabetic retinopathy. Am. J. Ophthalmol.113:36–38, 1992.Google Scholar
  41. 41.
    Terauchi, Y., H. Sakura, K. Yasuda, K. Iwamoto, N. Takahashi, K. Ito, H. Kasai, H. Suzuki, O. Ueda, and N. Kamada. Pancreatic-β cell specific targeted disruption of glucokinase gene: Diabetes mellitus due to defective insulin secretion to glucose. J. Biol. Chem.268:30253–30256, 1995.Google Scholar
  42. 42.
    Vanderkooi, J. M., G. Maniara, T. J. Green, and D. F. Wilson. An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem.252:5476–5482, 1987.Google Scholar
  43. 43.
    Vanzetta, I., and A. Grinvald. Commentary: Evidence and lack of evidence for the initial dip in the anesthetized rat: Implications for human functional brain imaging. Neuroimage13:959–967, 2001.Google Scholar
  44. 44.
    Vinogradov, S. A., L.-W. Lo, W. T. Jenkins, S. M. Evans, C. J. Koch, and D. F. Wilson. Noninvasive imaging of the distribution in oxygen in tissue using near-infrared phosphors. Biophys. J.70:1609–1617, 1996.Google Scholar
  45. 45.
    Wilson, D. F., A. Pastuszko, J. E. DiGiacomo, M. Pawlowski, R. Schneiderman, and M. Delivoria-Papadopoulos. Effect of hyperventilation on oxygenation of the brain cortex of newborn piglets. J. Appl. Physiol.70:2691–2696, 1991.Google Scholar
  46. 46.
    Withers, D. J., J. S. Gutierrez, H. Towery, D. J. Burks, J.-M. Ren, S. Previs, Y. Zhang, D. Bernal, S. Pons, G. I. Shulman, S. Bonner-Weir, and M. F. White. Disruption of the IRS-2 causes type 2 diabetes in mice. Nature (London)391:900–904, 1998.Google Scholar
  47. 47.
    Yu, D.-Y., S. J. Cringle, V. A. Alder, and E.-N. Su. Intraretinal oxygen distribution in the rat with graded systemic hyperoxia and hypercapnia. Invest. Ophthalmol. Visual Sci.40:2082–2087, 1999.Google Scholar
  48. 48.
    Zarbin, M. A.Age-related macular degeneration: Review of pathogenesis. Eur. J. Ophthalmol.8:199–206, 1998.Google Scholar

Copyright information

© Biomedical Engineering Society 2003

Authors and Affiliations

  • Ross D. Shonat
    • 1
  • Amanda C. Kight
    • 1
  1. 1.Department of Biomedical EngineeringWorcester Polytechnic InstituteWorcester

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