To date, brain imaging has largely relied on X-ray computed tomography and magnetic resonance angiography, with their limited spatial resolution and long scanning times. Fluorescence-based brain imaging in the visible and traditional near-infrared regions (400–900 nm) is an alternative, but at present it requires craniotomy, cranial windows and skull-thinning techniques, and the penetration depth is limited to 1–2 mm due to light scattering. Here, we report through-scalp and through-skull fluorescence imaging of mouse cerebral vasculature without craniotomy, utilizing the intrinsic photoluminescence of single-walled carbon nanotubes in the 1.3–1.4 μm near-infrared window (NIR-IIa window). Reduced photon scattering in this spectral region allows fluorescence imaging to a depth of >2 mm in mouse brain with sub-10-μm resolution. An imaging rate of ∼5.3 frames per second allows for dynamic recording of blood perfusion in the cerebral vessels with sufficient temporal resolution, providing real-time assessment of a blood flow anomaly in a mouse middle cerebral artery occlusion stroke model.
At a glance
- Heart disease and stroke statistics—2013 update—a report from the American Heart Association. Circulation 127, E6–E245 (2013). et al.
- Comparison of CT and CT angiography source images with diffusion-weighted imaging in patients with acute stroke within 6 hours after onset. Stroke 33, 2426–2432 (2002). et al.
- Digital reconstruction and morphometric analysis of human brain arterial vasculature from magnetic resonance angiography. Neuroimage 82, 170–181 (2013). et al.
- High-resolution structural and functional assessments of cerebral microvasculature using 3D gas ΔR2*-mMRA. PloS ONE 8, e78186 (2013). et al.
- First performance evaluation of a dual-source CT (DSCT) system. Eur. Radiol. 16, 256–268 (2006). et al.
- Dynamic changes in murine vessel geometry assessed by high-resolution magnetic resonance angiography: a 9.4 T study. J. Magn. Reson. Imaging 28, 637–645 (2008). et al.
- High resolution X-ray computed tomography: an emerging tool for small animal cancer research. Neoplasia 2, 62–70 (2000). , , , &
- In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photon. 7, 205–209 (2013).
- In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997). , , &
- Chronic optical access through a polished and reinforced thinned skull. Nature Methods 7, 981–984 (2010). et al.
- Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nature Protoc. 5, 201–208 (2010). , , , &
- Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor. Laboratory investigation. J. Neurosurg. 115, 1131–1138 (2011). et al.
- Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003). , &
- In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 16, 106014 (2011). , &
- 2007). & An Introduction to Biomedical Optics (Taylor & Francis,
- In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634 (2003).
- Bioimaging: second window for in vivo imaging. Nature Nanotech. 4, 710–711 (2009). , &
- M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors. Nano Lett. 12, 1176–1183 (2012). et al.
- A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nature Nanotech. 4, 773–780 (2009). et al.
- Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window. Proc. Natl Acad. Sci. USA 108, 8943–8948 (2011). , &
- Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates. Nature Commun. 3, 700 (2012). et al.
- Multifunctional in vivo vascular imaging using near-infrared II fluorescence. Nature Med. 18, 1841–1846 (2012). et al.
- Imaging depths of near-infrared quantum dots in first and second optical windows. Mol. Imaging 11, 338–352 (2012). et al.
- In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region. Angew. Chem. Int. Ed. 51, 9818–9821 (2012). et al.
- Rare-earth-doped biological composites as in vivo shortwave infrared reporters. Nature Commun. 4, 2199 (2013). et al.
- Biological imaging using nanoparticles of small organic molecules with fluorescence emission at wavelengths longer than 1000 nm. Angew. Chem. Int. Ed. 52, 13002–13006 (2013). et al.
- Facile synthesis of highly photoluminescent Ag2Se quantum dots as a new fluorescent probe in the second near-infrared window for in vivo imaging. Chem. Mater. 25, 2503–2509 (2013). et al.
- Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement. Circ. Cardiovasc. Imaging 7, 517–525 (2014). et al.
- Ultrafast fluorescence imaging in vivo with conjugated polymer fluorophores in the second near-infrared window. Nature Commun. 5, 4206 (2014). et al.
- Purification and characterization of single-wall carbon nanotubes (SWNTs) obtained from the gas-phase decomposition of CO (HiPco process). J. Phys. Chem. B 105, 8297–8301 (2001). et al.
- Band gap fluorescence from individual single-walled carbon nanotubes. Science 297, 593–596 (2002). et al.
- Structure-assigned optical spectra of single-walled carbon nanotubes. Science 298, 2361–2366 (2002). et al.
- Light scattering in Intralipid-10% in the wavelength range of 400–1100 nm. Appl. Opt. 30, 4507–4514 (1991). , , , &
- The near infrared absorption spectrum of liquid water. J. Opt. Soc. Am. 41, 302–304 (1951). &
- Optical properties of human cranial bone in the spectral range from 800 to 2000 nm. Saratov Fall Meeting 2005: Optical Technologies in Biophysics and Medicine VII 6163, 616310 (2006). , , &
- Optical properties of human skin, subcutaneous and mucous tissues in the wavelength range from 400 to 2000 nm. J. Phys. D 38, 2543–2555 (2005). , , &
- An evaluation of confocal versus conventional imaging of biological structures by fluorescence light-microscopy. J. Cell Biol. 105, 41–48 (1987). , &
- 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, 15741–15746 (1998). , , &
- Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cerebr. Blood Flow Metab. 32, 1277–1309 (2012). et al.
- In vivo measurements of blood flow and glial cell function with two-photon laser-scanning microscopy. Methods Enzymol. 444, 231–254 (2008). &
- Deep tissue two-photon microscopy. Nature Methods 2, 932–940 (2005). &
- Laser speckle contrast imaging of cerebral blood flow. Ann. Biomed. Eng. 40, 367–377 (2012).
- Laser speckle contrast imaging in biomedical optics. J. Biomed. Opt. 15, 011109 (2010). &
- Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nature Med. 15, 1219–1223 (2009). et al.
- Laser speckle contrast imaging: theory, instrumentation and applications. IEEE Rev. Biomed. Eng. 6, 99–110 (2013). , , &
- All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast. Nature Photon. 1, 526–530 (2007). &