Editor’s summary
Optical imaging of biological tissues is hindered by the scattering and, to a lesser extent, absorption of light that limits the penetration depth. Ou et al. addressed this problem through an approach that at first may seem counterintuitive: the introduction of highly absorbing molecules (see the Perspective by Rowlands and Gorecki). The authors show that the addition of common dye molecules that absorb in the near ultraviolet and blue regions improve optical transparency in nearby longer wavelengths. In essence, by causing sharp absorption in the blue region, the refractive index in the red part of the spectrum is increased without increasing absorption. The addition of tartrazine was able to make the skin of a live rodent temporarily transparent. —Marc S. Lavine
Structured Abstract
INTRODUCTION
A challenge in trying to image biological matter is that its complex structure causes opacity because of unwanted light scattering. This scattering results from refractive index mismatches among the components of biological tissues, limiting the penetration depth of optical imaging. The desire to see inside biological tissue and uncover the fundamental processes of life has spurred extensive research into deep-tissue optical imaging methods, such as two-photon microscopy, near-infrared-II fluorescence imaging, and optical tissue clearing. However, these methods either lack sufficient penetration depth and resolution or are unsuitable for living animals. Therefore, the ability to achieve optical transparency in live animals holds promise for transforming many optical imaging techniques.
RATIONALE
We hypothesized that strongly absorbing molecules can achieve optical transparency in live biological tissues. By applying the Lorentz oscillator model for the dielectric properties of tissue components and absorbing molecules, we predicted that dye molecules with sharp absorption resonances in the near-ultraviolet spectrum (300 to 400 nm) and blue region of the visible spectrum (400 to 500 nm) are effective in raising the real part of the refractive index of the aqueous medium at longer wavelengths when dissolved in water, which is in agreement with the Kramers-Kronig relations. As a result, water-soluble dyes can effectively reduce the RI contrast between water and lipids, leading to optical transparency of live biological tissues.
RESULTS
Following our theory, we found that an aqueous solution of a common food color approved by the US Food and Drug Administration, tartrazine, has the effect of reversibly making the skin, muscle, and connective tissues transparent in live rodents. We conducted experiments in both tissue-mimicking scattering hydrogels and ex vivo biological tissues. These tests confirmed the mechanism underlying our observations and showcased the achievable spatial resolution down to the micrometer level through millimeters of scattering medium once transparency is attained. By using absorbing dye molecules, we can transform the typically opaque abdomen of a live mouse into a transparent medium. This “transparent abdomen” allows for direct visualization of fluorescent protein–labeled enteric neurons, capturing their movements that mirror the underlying gut motility in live mice. This enabled us to generate time-evolving maps that depict mouse gut motility and the diversity of movement patterns. To demonstrate the generalizability of this approach, we also applied dye solutions topically to the scalp of a mouse head for visualizing cerebral blood vessels and to the mouse hindlimb for high-resolution microscopic imaging of muscle sarcomeres.
CONCLUSION
Overall, we report the counterintuitive observation that strongly absorbing molecules can achieve optical transparency in live animals. The Lorentz oscillator model, which underlies this unusual observation, predicts that molecules with low resonance frequencies (long absorption wavelengths), sharp absorption peaks, and rich delocalized electrons are more effective candidates at raising the refractive index of the aqueous medium than are conventional optical clearing agents. Our approach also presents opportunities for visualizing the structure, activity, and functions of deep-seated tissues and organs without the need for surgical removal or the replacement of overlying tissues with transparent windows. Some limitations remain for this method, including reduced but not eliminated scattering owing to the challenge of matching refractive indices in heterogeneous tissues and achievable penetration depth depending on the diffusion of absorbing molecules.
