Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Attwell, D. et al. Nature 468, 232–243 (2010).
Kleinfeld, D. et al. Front. Neuroenergetics 3, 1 (2011).
Iadecola, C. & Nedergaard, M. Nat. Neurosci. 10, 1369–1376 (2007).
O'Kusky, J. & Colonnier, M. J. Comp. Neurol. 210, 278–290 (1982).
Ohki, K. et al. Nature 442, 925–928 (2006).
Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Nature 433, 597–603 (2005).
Kara, P. & Boyd, J.D. Nature 458, 627–631 (2009).
Logothetis, N.K. Nature 453, 869–878 (2008).
Berlier, J.E. et al. J. Histochem. Cytochem. 51, 1699–1712 (2003).
Drew, P.J. et al. Nat. Methods 7, 981–984 (2010).
Tian, P.F. et al. Proc. Natl. Acad. Sci. USA 107, 15246–15251 (2010).
Drew, P.J., Shih, A.Y. & Kleinfeld, D. Proc. Natl. Acad. Sci. USA 108, 8473–8478 (2011).
Zipfel, W.R. et al. Proc. Natl. Acad. Sci. USA 100, 7075–7080 (2003).
Kansui, Y., Garland, C.J. & Dora, K.A. Cell Calcium 44, 135–146 (2008).
Clifford, P.S. et al. Arterioscler. Thromb. Vasc. Biol. 31, 2889–2896 (2011).
Drew, P.J., Blinder, P., Cauwenberghs, G., Shih, A.Y. & Kleinfeld, D. J. Comput. Neurosci. 29, 5–11 (2010).
Carlson, T.R. et al. Proc. Natl. Acad. Sci. USA 102, 9884–9889 (2005).
Li, Y., Lu, Z., Keogh, C.L., Yu, S.P. & Wei, L. J. Cereb. Blood Flow Metab. 27, 1043–1054 (2007).
Svedin, P., Hagberg, H., Savman, K., Zhu, C. & Mallard, C. J. Neurosci. 27, 1511–1518 (2007).
Bell, R.D. et al. Neuron 68, 409–427 (2010).
Bolte, S. & Cordelieres, F.P. J. Microsc. 224, 213–232 (2006).
Zinchuk, V. & Grossenbacher-Zinchuk, O. Prog. Histochem. Cytochem. 44, 125–172 (2009).
Acknowledgements
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
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
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.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–20, Supplementary Notes 1–2 (PDF 9873 kb)
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). (MOV 1087 kb)
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). (MOV 2505 kb)
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). (MOV 7878 kb)
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. (MOV 306 kb)
Rights and permissions
About this article
Cite this article
Shen, Z., Lu, Z., Chhatbar, P. et al. An artery-specific fluorescent dye for studying neurovascular coupling. Nat Methods 9, 273–276 (2012). https://doi.org/10.1038/nmeth.1857
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nmeth.1857
This article is cited by
-
Surmounting photon limits and motion artifacts for biological dynamics imaging via dual-perspective self-supervised learning
PhotoniX (2024)
-
Fluorescence-amplified nanocrystals in the second near-infrared window for in vivo real-time dynamic multiplexed imaging
Nature Nanotechnology (2023)
-
Adaptive optical microscopy via virtual-imaging-assisted wavefront sensing for high-resolution tissue imaging
PhotoniX (2022)
-
Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels
Nature Methods (2022)
-
Astrocyte plasticity in mice ensures continued endfoot coverage of cerebral blood vessels following injury and declines with age
Nature Communications (2022)