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An artery-specific fluorescent dye for studying neurovascular coupling

Nature Methods volume 9, pages 273276 (2012) | Download Citation


We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus–evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.

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We thank W. Argraves (Medical University of South Carolina) for mouse aorta samples; J. Horton (University of California at San Francisco) for macaque monkey brain tissue; C. Beeson and Y. Peterson for modeling the structural overlap of various Alexa dyes; P. Bell for dissecting mouse kidneys; D. Leopold and A. Silva for discussion on MRI applications; R. Gourdie for discussion of Alexa Fluor 633 binding sites on artery walls; E. Vought for creating illustrations; P. Mulholland for help with using Imaris software; M. Levy and A. Schramm for comments on the manuscript. This work was supported by grants from the US National Eye Institute (R01EY017925 and R21EY020985) and funds from the Medical University of South Carolina to P.K.

Author information

Author notes

    • Zhiming Shen
    •  & Zhongyang Lu

    These authors contributed equally to this work.


  1. Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA.

    • Zhiming Shen
    • , Zhongyang Lu
    • , Pratik Y Chhatbar
    • , Philip O'Herron
    •  & Prakash Kara


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P.K. conceived and designed the study, except the head angle with dip artifact experiments (Z.S.). All authors performed in vivo vessel imaging experiments. Z.S. did in vivo calcium imaging and head-angle experiments. Z.L. did histology on brain and femoral vessel tissue samples. P.K. did histology of mouse aorta, mouse kidney and human aorta tissues. P.K., Z.S., P.Y.C. and P.O. analyzed the in vivo vessel dilation and velocity data. P.K. and Z.S. analyzed the in vivo calcium imaging and single-cell electroporation data. All authors discussed results. P.K. wrote the manuscript.

Competing interests

The Medical University of South Carolina Foundation for Research Development has filed a provisional patent application US 61/626,314 that names P.K. as an inventor and encompasses aspects of the technology described in this paper.

Corresponding author

Correspondence to Prakash Kara.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–20, Supplementary Notes 1–2


  1. 1.

    Supplementary Video 1

    Delay between the filling of arteries and veins after intravenous dye injection confirm artery specificity of Alexa Fluor 633 labeling in vivo. Time-lapse epifluorescence imaging of cortical vessels immediately after fluorescein dextran was injected intravenously in the femoral vein. The entire x-y field of view shown represents a region of 1.87 mm × 1.87 mm in the cat visual cortex. Because the dye must pass from the femoral (leg) vein through the heart to get to the cerebral cortex, the arteries must label first. Images were collected using a monochrome CCD camera (see Supplementary Fig. 1).

  2. 2.

    Supplementary Video 2

    Small cortical surface arterioles were labeled in vivo by Alexa Fluor 633, whereas venules were not. High-optical-zoom two-photon z stack from the rat visual cortex. The x-y field of view shown represents a region of 109 μm × 109 μm. The 18 z steps of the movie span 34 μm. The lumen of the vessels is visible (green) after an intravenous injection of fluorescein dextran. The vessel walls of a primary arteriole and daughter branchlet are well labeled by an intravenous injection of Alexa Fluor 633 (red) whereas a nearby venule is unlabeled (see Supplementary Fig. 2).

  3. 3.

    Supplementary Video 3

    Cortical surface and penetrating arteriole ≥15 μm diameter were labeled in vivo by Alexa Fluor 633 whereas veins and microvessels were not. A 360° rotation view of a 220 μm × 234 μm × 123 μm two-photon volume from mouse visual cortex. All vessels are visible (green) after an intravenous injection of fluorescein dextran but only the surface and penetrating arterioles are labeled by Alexa Fluor 633 (Supplementary Fig. 5c,d).

  4. 4.

    Supplementary Video 4

    Raw two-photon imaging frames showing an increase in arteriole vessel diameter upon sensory visual stimulation in vivo. Raw data frames (left) and frame-locked presentation (right) of blank (gray) and drifting grating visual stimuli. Data are shown for 15 frames: 5 frames blank, followed by 5 frames of visual stimulation and then 5 frames blank. The duration of each frame was 1.48 s. The entire x-y field of view shown in the 2 two-photon imaging frames represents a region of 553 μm × 553 μm from rat visual cortex close to the pial surface.

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