Dynamic Two-Photon Imaging of Cerebral Microcirculation Using Fluorescently Labeled Red Blood Cells and Plasma

  • Kazuto Masamoto
  • Hiroshi Kawaguchi
  • Hiroshi Ito
  • Iwao Kanno
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 765)


To explore the spatiotemporal dynamics of red blood cells (RBCs) and plasma flow in three-dimensional (3D) microvascular networks of the cerebral cortex, we performed two-photon microscopic imaging of the cortical microvasculature in genetically engineered rats in which the RBCs endogenously express green fluorescent protein (GFP). Water-soluble quantum dots (Qdots) were injected intravenously into the animals to label the plasma, and concurrent imaging was performed for GFP-RBCs and Qdot plasma. The RBC and plasma distributions were compared between resting state and forepaw stimulation-induced neural activation. The RBC and plasma images showed detectable signals up to a depth of 0.4 and 0.6 mm from the cortical surface, respectively. A thicker plasma layer (2–5 μm) was seen in venous vessels relative to the arterial vessels. In response to neural activation, the RBCs were redistributed among the parenchymal capillary networks. In addition, individual capillaries showed a variable ratio of RBC and plasma distributions before and after activation, indicative of dynamic changes of hematocrit in single capillaries. These results demonstrate that this transgenic animal model may be useful in further investigating the mechanism that controls dynamic RBC flow in single capillaries and among multiple capillary networks of the cerebral microcirculation.


Brain microcirculation Functional imaging Oxygen demand and supply Somatosensory cortex 



The authors thank Dr. Junko Taniguchi for help with the experiments. This work was partly supported by Special Coordination Funds for Promoting Science and Technology (K.M.).


  1. 1.
    Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76CrossRefGoogle Scholar
  2. 2.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Park SH, Masamoto K, Hendrich K et al (2008) Imaging brain vasculature with BOLD microscopy: MR detection limits determined by in vivo two-photon microscopy. Magn Reson Med 59:855–865CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yoshihara K, Takuwa H, Kanno I et al (2012) 3D analysis of intracortical microvasculature during chronic hypoxia in mouse brain. Adv Exp Med Biol 765: 357–363Google Scholar
  5. 5.
    Kleinfeld D, Mitra PP, Helmchen F et al (1998) Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc Natl Acad Sci USA 95:15741–15746CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schaffer CB, Friedman B, Nishimura N et al (2006) Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLoS Biol 4:e22CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dobbe JG, Streekstra GJ, Atasever B et al (2008) Measurement of functional microcirculatory geometry and velocity distributions using automated image analysis. Med Biol Eng Comput 46:659–670CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Drew PJ, Blinder P, Cauwenberghs G et al (2010) Rapid determination of particle velocity from space-time images using the Radon transform. J Comput Neurosci 29:5–11CrossRefPubMedGoogle Scholar
  10. 10.
    Kamoun WS, Chae SS, Lacorre DA et al (2010) Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat Methods 7:655–660CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Autio J, Kawaguchi H, Saito S, Aoki I, Obata T, Masamoto K, Kanno I (2011) Spatial frequency-based analysis of mean red blood cell speed in single microvessels: investigation of microvascular perfusion in rat cerebral cortex. PLoS One 6:e24056CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tomita M, Osada T, Schiszler I et al (2008) Automated method for tracking vast numbers of FITC-labeled RBCs in microvessels of rat brain in vivo using a high-speed confocal microscope system. Microcirculation 15:163–174CrossRefPubMedGoogle Scholar
  13. 13.
    Unekawa M, Tomita M, Tomita Y et al (2010) RBC velocities in single capillaries of mouse and rat brains are the same, despite 10-fold difference in body size. Brain Res 1320:69–73CrossRefPubMedGoogle Scholar
  14. 14.
    Tomita M, Tomita Y, Unekawa M et al (2011) Oscillating neuro-capillary coupling during cortical spreading depression as observed by tracking of FITC-labeled RBCs in single capillaries. Neuroimage 56:1001–1010CrossRefPubMedGoogle Scholar
  15. 15.
    Masamoto K, Obata T, Kanno I (2010) Intracortical microcirculatory change induced by anesthesia in rat somatosensory cortex. Adv Exp Med Biol 662:57–61CrossRefPubMedGoogle Scholar
  16. 16.
    Masamoto K, Kim T, Fukuda M et al (2007) Relationship between neural, vascular, and BOLD signals in isoflurane-anesthetized rat somatosensory cortex. Cereb Cortex 17:942–950CrossRefPubMedGoogle Scholar
  17. 17.
    Kim T, Masamoto K, Fukuda M et al (2010) Frequency-dependent neural activity, CBF, and BOLD fMRI to somatosensory stimuli in isoflurane-anesthetized rats. Neuroimage 52:224–233CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Resch-Genger U, Grabolle M, Cavaliere-Jaricot S et al (2008) Quantum dots versus organic dyes as fluorescent labels. Nat Methods 5:763–775CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kazuto Masamoto
    • 1
    • 2
  • Hiroshi Kawaguchi
    • 2
  • Hiroshi Ito
    • 2
  • Iwao Kanno
    • 2
  1. 1.Center for Frontier Science and EngineeringUniversity of Electro-CommunicationsChofuJapan
  2. 2.Molecular Imaging CenterNational Institute of Radiological SciencesChibaJapan

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