A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle

Journal name:
Nature Medicine
Year published:
Published online


The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging–photoacoustic imaging–Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality–nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.

At a glance


  1. Triple-modality MPR concept.
    Figure 1: Triple-modality MPR concept.

    MPRs are injected intravenously into a mouse bearing an orthotopic brain tumor (top). As the nanoparticles circulate in the bloodstream, they diffuse through the disrupted blood-brain barrier and are then sequestered and retained by the tumor. The MPRs are too large to cross the intact blood-brain barrier and, therefore, cannot accumulate in healthy brain. The concept of proposed eventual clinical use (bottom). Detectability of MPRs by MRI allows for preoperative detection and surgical planning. Because of the retention of the probe, only one injection is necessary, and the probe can be detected in the tumor during surgery several days later. Photoacoustic imaging, with its relatively high resolution and deep tissue penetration, is then able to guide bulk tumor resection intraoperatively. Raman imaging, with its ultrahigh sensitivity and spatial resolution, can then be used to remove any residual microscopic tumor burden. The resected specimen can subsequently be examined using a Raman probe ex vivo to verify clear tumor margins.

  2. Characterization of the MPRs.
    Figure 2: Characterization of the MPRs.

    (a) Simplified diagram of the MPR. A 60-nm gold core is surrounded by a thin Raman-active layer that is protected by a 30-nm silica coating. The silica coating was further functionalized with maleimide-DOTA-Gd, which was conjugated to the thiol (SH) group on the silica. (b) Transmission electron microscopy images of the MPRs. (c) Particle relaxivity derived from T1 maps of probe dilutions in MRI phantoms. Data represent the mean of two separate phantoms containing separate probe conjugations (error bars (s.e.m.) indicate batch-to-batch variation). The inset shows a T1 map of an MRI phantom containing MPRs at concentrations ranging from 3.2 nM (1) to 25 pM (8). (d) Optical absorbance of the MPRs. (e) Raman spectrum of the MPRs, with characteristic peaks at 1,021, 1,204, 1,340, 1,614 and 1,638 cm−1. (f,g) During 30 min of continuous laser irradiation, the optical absorbance (f) and the Raman signal (g) remained constant. AU, arbitrary units.

  3. Triple-modality detection of brain tumors in living mice with MPRs.
    Figure 3: Triple-modality detection of brain tumors in living mice with MPRs.

    (a) Two-dimensional axial MRI, photoacoustic and Raman images. The post-injection images of all three modalities showed clear tumor visualization (dashed boxes outline the imaged area). (b) A three-dimensional (3D) rendering of magnetic resonance images with the tumor segmented (red; top), an overlay of the three-dimensional photoacoustic images (green) over the MRI (middle) and an overlay of MRI, the segmented tumor and the photoacoustic images (bottom) showing good colocalization of the photoacoustic signal with the tumor. (c) Quantification of the signals in the tumor showing a significant increase in the MRI, photoacoustic and Raman signals after as compared to before the injection. n = 4 mice. Data represent mean ± s.e.m. ***P < 0.001, **P < 0.01 (one-sided Student's t test.) AU, arbitrary units.

  4. Histological validation.
    Figure 4: Histological validation.

    Ten-micrometer frozen sections from the margin of an eGFP+U87MG brain tumor stained for eGFP (green) to visualize the tumor margins and CD11b (red) to visualize glial cells and examined using laser scanning confocal microscopy (top). A 50-μm adjacent slice examined using Raman microscopy to visualize the distribution of the MPRs (bottom). The Raman signal corresponding to the eGFP+ cells indicates the presence of the probe in the tumor but not in the adjacent healthy tissue. The Raman signal (red) was scaled from 0 to 100 AU. The boxes are not drawn to scale. STEM images verified the presence of MPRs in the brain tissue, whereas no MPRs were seen in the healthy brain tissue. A three-dimensional STEM rendering of MPRs in brain tumor is provided in Supplementary Video 1.

  5. Raman-guided intraoperative surgery using MPRs.
    Figure 5: Raman-guided intraoperative surgery using MPRs.

    (a,b) Living tumor-bearing mice (n = 3) underwent craniotomy under general anesthesia. Quarters of the tumor were then sequentially removed (as illustrated in the photographs, a), and intraoperative Raman imaging was performed after each resection step (b) until the entire tumor had been removed, as assessed by visual inspection. After the gross removal of the tumor, several small foci of Raman signal were found in the resection bed (outlined by the dashed white square; some Raman images are smaller than the image frame). The Raman color scale is shown in red from −40 dB to 0 dB. (c) A subsequent histological analysis of sections from these foci showed an infiltrative pattern of the tumor in this location, forming finger-like protrusions extending into the surrounding brain tissue. As shown in the Raman microscopy image (right), a Raman signal was observed within these protrusions, indicating the selective presence of MPRs. The box is not drawn to scale. The Raman signal is shown in a linear red color scale.


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Author information

  1. These authors contributed equally to this work.

    • Moritz F Kircher &
    • Adam de la Zerda


  1. Molecular Imaging Program at Stanford, Department of Radiology, Stanford University, Stanford, California, USA.

    • Moritz F Kircher,
    • Adam de la Zerda,
    • Jesse V Jokerst,
    • Cristina L Zavaleta,
    • Erik Mittra,
    • Frezghi Habte &
    • Sanjiv S Gambhir
  2. Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Moritz F Kircher &
    • Ruimin Huang
  3. Department of Radiology, Weill Cornell Medical College, New York, New York, USA.

    • Moritz F Kircher
  4. Brain Tumor Center, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Moritz F Kircher,
    • Ken Pitter,
    • Ruimin Huang,
    • Cameron W Brennan &
    • Eric C Holland
  5. Department of Electrical Engineering, Stanford University, Stanford, California, USA.

    • Adam de la Zerda
  6. Department of Materials Science & Engineering, Stanford University, Stanford, California, USA.

    • Paul J Kempen,
    • Robert Sinclair &
    • Sanjiv S Gambhir
  7. Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Ken Pitter &
    • Eric C Holland
  8. Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Ruimin Huang,
    • Ingo K Mellinghoff &
    • Eric C Holland
  9. Human Oncology & Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Carl Campos,
    • Cameron W Brennan &
    • Ingo K Mellinghoff
  10. Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Cameron W Brennan &
    • Eric C Holland
  11. Department of Neurosurgery, Weill Cornell Medical College, New York, New York, USA.

    • Cameron W Brennan &
    • Eric C Holland
  12. Department of Pharmacology, Weill Cornell Medical College, New York, New York, USA.

    • Ingo K Mellinghoff
  13. Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Eric C Holland
  14. Department of Bioengineering and Bio-X Program, Stanford University, Stanford, California, USA.

    • Sanjiv S Gambhir


M.F.K. co-initiated the project, designed the research, synthesized and characterized MPR nanoparticles, performed MRI, Raman, photoacoustic and histology experiments, analyzed data and wrote the manuscript. A.d.l.Z. modified the photoacoustic system, designed and performed photoacoustic experiments, analyzed data and wrote the manuscript. J.V.J. synthesized and characterized MPR nanoparticles. C.L.Z. designed, performed and analyzed Raman experiments and edited the paper. P.J.K. and R.S. performed and analyzed the electron microscopy experiments. K.P. performed immunohistochemistry. F.H. helped create three-dimensional renderings. E.M., M.F.K., K.P., R.H., C.C., C.W.B., I.K.M. and E.C.H. provided mouse models. S.S.G. co-initiated the project, designed the research, analyzed data, supervised and coordinated all investigators for the project and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (5M)

    Supplementary Figures 1–14, Supplementary Discussion and Supplementary Methods


  1. Supplementary Video 1 (10M)

    Three-dimensional STEM rendering of MPR nanoparticles in U87MG tumor

Additional data