Abstract
Optical imaging provides real-time visualization of tissues and cells at high spatial and temporal resolutions through techniques such as fluorescence microscopy, optical coherence tomography and photoacoustic imaging. However, overcoming light scattering, caused by mismatches in the refractive indices of tissue components such as water and lipids, still represents a major challenge, particularly when imaging through the thicker biological tissues of living animals. Despite advances in deep-tissue imaging, many optical methods struggle to achieve diffraction-limited resolution at depth or are unsuitable for use in live animals. Here we introduce a noninvasive approach to achieving transient and reversible optical transparency in live mice using absorbing dye molecules, using tartrazine as a representative example. Rooted in the fundamental physics of light–matter interactions, this approach enables reversible optical transparency in live animals and can be further applied ex vivo in freshly dissected tissues. In this Protocol, we detail the procedures for visualizing in vivo internal organs and muscle sarcomeres in the mouse abdomen and hindlimb through their respective transparency windows, showcasing a versatile approach for a variety of optical imaging applications in live animals. The entire protocol for an in vivo application can be implemented in just over 2 weeks by users with expertise in optical imaging and animal handling.
Key points
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The procedure includes the preparation and application of absorbing molecules to create transient optical transparency windows ex vivo and in vivo, enabling the imaging of deep tissue structures and biological processes at macroscopic and microscopic scales.
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Alternative methods for deep tissue biological imaging, such as microendoscopes and cranial or abdominal windows, are invasive and can cause damage and disruption to the natural composition and activity of the tissue.
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Data availability
All relevant source data supporting this study are included in the paper and available in the accompanying source data files. Characterized and quality-controlled tartrazine solutions are available to other research laboratories upon request. Source data are provided with this paper.
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Acknowledgements
Ellipsometry characterizations were performed at the Stanford Nano Shared Facilities, supported by the National Science Foundation (grant no. ECCS-1542152). Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. In vivo multiphoton imaging experiments were performed at Stanford Wu Tsai Neuroscience Microscopy Service, with help from G. Wang. G.H. acknowledges three awards from the NIH (grant nos. 5R00AG056636-04, 1R34NS127103-01 and R01NS126076), an NSF CAREER award (grant no. 2045120), an NSF EAGER award (grant no. 2217582), a Rita Allen Foundation Scholars Award, a Beckman Technology Development Grant, a grant from the Focused Ultrasound Foundation, a gift from the Spinal Muscular Atrophy Foundation, gifts from the Pinetops Foundation, two seed grants from the Wu Tsai Neurosciences Institute, two seed grants from the Bio-X Initiative of Stanford University and a teacher-scholar award from the Camille and Henry Dreyfus Foundation. M.L.B. acknowledges a grant from the Air Force Office of Scientific Research (grant no. FA9550-21-1-0312). C.H.C.K. acknowledges the National Science Foundation Graduate Research Fellowships program (grant no. 1656518) and the Wu Tsai Neuroscience NeuroTech Training program. E.L.S. acknowledges a TIME fellowship. L.Z. acknowledges a Knight-Hennessy Fellowship. H.C. acknowledges a Stanford Interdisciplinary Graduate Fellowship.
Author information
Authors and Affiliations
Contributions
G.H. and M.L.B. contributed ideas and designed research. C.H.C.K., E.L.S. and G.H. made tartrazine solutions and characterized their optical properties. C.H.C.K., E.L.S. and G.H. prepared mouse skin samples and performed ex vivo transparency experiments. C.H.C.K., S.Z., Z.L., L.Z. and G.H. prepared live mice and performed in vivo transparency experiments. M.C., X.C. and C.W. prepared bioadhesive hydrogels. C.H.C.K., E.L.S., M.L.B. and G.H. wrote the paper.
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G.H., M.L.B. and S.Z. are inventors on patent application (patent no. WO2023122534A1) submitted by Stanford University that covers the principles of achieving optical transparency by applying the Kramers–Kronig relations.
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Nature Protocols thanks Arthur Petusseau, Myunghwan Choi, Conor Evans and Wonsang Hwang for their contribution to the peer review of this work.
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Key references
Ou, Z. et al. Science 385, eadm6869 (2024): https://doi.org/10.1126/science.adm6869
Miller, D. A. et al. Optica 12, 24–30 (2025): https://doi.org/10.1364/OPTICA.546779
Extended data
Extended Data Fig. 1 Instability of tartrazine solutions.
a, Pure tartrazine solution after 96 h at room temperature, featuring complete precipitation. b, Pure tartrazine solution after complete precipitation, followed by 10 min in an 80 °C oven to redissolve the precipitates. c, Tartrazine/PVA solutions after 96 h at room temperature, featuring complete precipitation. d, Tartrazine/PVA solution after complete precipitation, followed by 10 min in an 80 °C oven to redissolve the precipitates.
Extended Data Fig. 2 The transparency window in the mouse abdomen appears orange to red after the skin has fully absorbed the tartrazine molecules.
a, An orange-to-red transparency window in the abdomen of a 23-day-old female C57Bl/6J mouse weighing 9.8 g. Excess tartrazine was removed before taking the photo. Note the orange-red color in this video comes from the tartrazine dye, not blood. b, A close-up view of the transparency window is shown by pressing a coverslip onto the abdomen. c, A white dashed line is overlaid on the photo to mark the red/yellow boundary in the skin. Scale bars: 1 cm.
Extended Data Fig. 3 Dissected skin after sufficient absorption of tartrazine becomes red and transparent.
In this example, a 22-day-old female C57Bl/6J mouse weighing 10.5 g was used. Tartrazine was applied for 10 min, and excess tartrazine was removed before the skin was dissected.
Supplementary information
Supplementary Video 1
Achieving and reversing optical transparency in a live mouse. This video shows the entire process of depilation, exfoliation, tartrazine application, imaging and transparency reversal.
Supplementary Video 2
A clear boundary can be observed between the transparent (red) and opaque (yellow) regions of the skin on the mouse abdomen. After the skin has fully absorbed tartrazine molecules, it appears red and translucent, while depilated areas that have been in contact with the dye but have not absorbed sufficient amounts still appear yellow once the excess dye is removed. Note that when the skin on the left side of the mouse abdomen is moved by dragging with fingers, the underlying gut structures become masked or revealed, depending on the transparency of the skin above.
Supplementary Video 3
Reversing optical transparency in a live mouse. This video begins with a live mouse featuring a transparency window in the abdomen, enabling imaging through the skin. It then demonstrates the process of extracting tartrazine from the skin to reverse the transparency effect, applying a hydrogel to maintain skin hydration after reversal and securing the hydrogel with medical tape to protect it before the mouse is recovered from anesthesia.
Source data
Source Data Fig. 3
Baseline-subtracted UV-visible absorption spectrum of the 0.62 mM tartrazine solution with an optical path length of 1 mm.
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Keck, C.H.C., Schmidt, E.L., Zhao, S. et al. Achieving transient and reversible optical transparency in live mice with tartrazine. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01187-z
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DOI: https://doi.org/10.1038/s41596-025-01187-z
