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Volumetric real-time multispectral optoacoustic tomography of biomarkers


Multispectral optoacoustic tomography (MSOT) has recently been developed to enable visualization of optical contrast and tissue biomarkers, with resolution and speed representative of ultrasound. In the implementation described here, MSOT enables operation in real-time mode by capturing single cross-sectional images in <1 ms from living small animals (e.g., mice) and other tissues of similar dimensions. At the core of the method is illumination of the object using multiple wavelengths in order to resolve spectrally distinct biomarkers over background tissue chromophores. The system allows horizontal placement of a mouse in the imaging chamber and three-dimensional scanning of the entire body without the need to immerse the mouse in water. Here we provide a detailed description of the MSOT scanner components, system calibration, selection of image reconstruction algorithms and animal handling. Overall, the entire protocol can be completed within 15–30 min for acquisition of a whole-body multispectral data set from a living mouse.

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Figure 1: MSOT system and its key components.
Figure 2: Characterization of the ultrasound detection array.
Figure 3: Flow chart of the multispectral imaging loop.
Figure 4: Single-wavelength optoacoustic images of mouse anatomy taken at 750 nm.
Figure 5: Visualization of contrast agent distribution ex vivo.
Figure 6: Consecutive frames of a video showing the respiratory movements of a 3-week-old CD1 mouse over a duration of 9 s.
Figure 7: In vivo tracking of systemic ICG injection into a mouse.


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D.R. acknowledges support from the German Research Foundation (DFG) Research Grant (RA 1848/1) and the European Research Council (ERC) Starting Grant. V.N. acknowledges support from the ERC Advanced Investigator Award and the German Ministry of Education (BMBF) Innovation in Medicine Award.

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Authors and Affiliations



D.R. and A.B. developed the imaging system. A.B. designed and performed the experiments. D.R. and V.N. provided conceptual input and supervised the research. All authors contributed to writing the manuscript.

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Correspondence to Daniel Razansky.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Real-time video showing mouse motion in the abdomen area due to respiration and other effects. (MOV 9761 kb)

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Razansky, D., Buehler, A. & Ntziachristos, V. Volumetric real-time multispectral optoacoustic tomography of biomarkers. Nat Protoc 6, 1121–1129 (2011).

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