In vivo optical clearing of the mouse brain
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eLife Assessment
An in vivo imaging medium raises the refractive index of the fluid surrounding cells in the brain, rendering living tissue more accessible to light for in vivo experimentation. This important approach enables imaging neurons more brightly and at greater depth than in untreated tissue. Solid evidence, consisting of both optical and electrophysiological neural activity recordings in brain slices and in living mice, demonstrate a clearing effect, albeit subtle, and not without a significant impact on mice behavior, which limits the use of the proposed method. This work will be of relevance to any optical studies of in vivo tissue.
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Abstract
Light microscopy is one of the most powerful tools for understanding living systems, but the opacity of tissue prevents visualization of all but superficial layers. Several methods to clarify tissue have been developed, but most require fixed specimens. To address the challenge of improving resolution in functioning neuronal circuits, we developed a biocompatible clearing agent, iodixanol-ACSF, which is capable of increasing the transparency of living neuronal tissue. Brain-cleared mice were motile and unimpaired on a variety of behavioral tasks, and extracellular recordings showed that many cellular and circuit phenomena were well-preserved. In live iodixanol-ACSF cleared mouse brain tissue, both transmission and cellular-resolution fluorescence microscopy indicate improvements of 150-200% in penetration depth with one-third to one-half the laser intensity when compared to untreated tissue. Our results show that iodixanol-ACSF clearing will enable deeper imaging and extend our understanding of neuronal circuit function.
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eLife Assessment
An in vivo imaging medium raises the refractive index of the fluid surrounding cells in the brain, rendering living tissue more accessible to light for in vivo experimentation. This important approach enables imaging neurons more brightly and at greater depth than in untreated tissue. Solid evidence, consisting of both optical and electrophysiological neural activity recordings in brain slices and in living mice, demonstrate a clearing effect, albeit subtle, and not without a significant impact on mice behavior, which limits the use of the proposed method. This work will be of relevance to any optical studies of in vivo tissue.
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Reviewer #1 (Public review):
Summary:
Scattering of light sets the practical limits on optical imaging depth in living brain tissue. It arises from a refractive index mismatch between the fluid surrounding cells (~1.34) and the structures embedded in it (~1.35-1.43), and, unlike optical aberration, it cannot be effectively pre-compensated by adaptive optics. The authors present an approach that reduces scattering at its source, raising the refractive index of the extracellular fluid with a medium built around iodixanol, a non-ionic iodinated compound already approved for intravenous use in humans, formulated to remain isosmotic with standard artificial cerebrospinal fluid. They then evaluate it on three counts: how much it clears tissue, whether animals tolerate it, and whether circuit activity survives it. The paper also reports the …
Reviewer #1 (Public review):
Summary:
Scattering of light sets the practical limits on optical imaging depth in living brain tissue. It arises from a refractive index mismatch between the fluid surrounding cells (~1.34) and the structures embedded in it (~1.35-1.43), and, unlike optical aberration, it cannot be effectively pre-compensated by adaptive optics. The authors present an approach that reduces scattering at its source, raising the refractive index of the extracellular fluid with a medium built around iodixanol, a non-ionic iodinated compound already approved for intravenous use in humans, formulated to remain isosmotic with standard artificial cerebrospinal fluid. They then evaluate it on three counts: how much it clears tissue, whether animals tolerate it, and whether circuit activity survives it. The paper also reports the limits of the approach, documenting reduced spontaneous exploratory behavior in treated animals.
Strengths:
The optical characterization in brain slices is rigorous. The authors quantify clearing with the modulation transfer function, show that the effect reverses on washout, show that it does not develop in untreated tissue over matched durations, and repeat the measurement with a low-numerical-aperture objective specifically to separate reduced scattering from corrected aberration. Together these establish the core optical claim that the treatment reduces scattering, and not merely that it corrects aberration. The behavioral work is thorough: blinded testing across a series of behaviors, along with appropriate controls including a non-surgical group to separate the effects of surgery from those of the reagents, and computed tomography to verify that each tested animal received the compound. The authors also report the limitations explicitly, identifying a dissociation between motivated tasks (largely unaffected) and spontaneous exploration (substantially reduced). The retinal recordings are a well-chosen functional assay, and benchmarking against 2% isoflurane is useful.
Weaknesses:
(1) It is not clear how the reported improvement of 150-200% in penetration depth was derived, or how penetration depth was operationally defined. The authors should clarify both.
(2) Depth in the intact animal could be characterized more fully. Figure 5b plots contrast ratio against depth to roughly 450 µm, and Figure 6f shows a single plane at 430 µm, but neither gives a systematic depth series or a criterion-based depth limit. The contrast gain in Figure 5b also appears scattered rather than following a trend with depth, which deserves comment; limited penetration of the agent into deeper tissue may contribute.
(3) The quantitative analysis in Figure 6 could be strengthened. It currently shows traces from a single cell, with no population-level comparison of event rate, amplitude, decay kinetics, or pairwise correlation. As it stands, the figure supports the optical claim that the same cells are brighter, but not the physiological claim made alongside it.
(4) The authors may, if they choose, strengthen the biocompatibility analysis by adding histology for gliosis and cell death, measures of intracranial pressure or edema, and quantification of tissue volume change.
(5) The abstract overstates the behavioral result, stating that "Brain-cleared mice were motile and unimpaired on a variety of behavioral tasks." The animals were indeed motile and completed every task, and the motivated-task result is fair, but "unimpaired" is not the right summary of the spontaneous-activity data the authors themselves collected.
(6) The interpretive consequences of the behavioral findings could be discussed in more detail. A reduction in spontaneous locomotion and exploration of this magnitude would likely confound studies of spontaneous behavior, exploratory drive, or anxiety-like measures. Readers would benefit from explicit guidance on which designs are compatible with in vivo clearing. This is an argument for stating the method's domain of validity, not against the method.
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Reviewer #2 (Public review):
Summary:
The authors attack an important and challenging problem on how to make living tissues (especially mammalian brain) more transparent for light microscopy, while minimizing or eliminating the toxic side effects of such intervention. A few groundbreaking publications have advanced this field recently, eg. (Ou et al., Science, 2024, Inagaki et al., Nat. Meth. 2026). In this work, the authors apply iodixanol to increase the refractive index (RI) of ACSF, thereby making brain tissues slightly more transparent while minimizing the osmotic stress associated with such intervention.
Strengths:
The approach is methodologically valid, and the authors carefully tested both optical and behavior effects of I-ACSF injection in mice.
Weaknesses:
(1) The authors do not cite a recent key paper in the field of in vivo …
Reviewer #2 (Public review):
Summary:
The authors attack an important and challenging problem on how to make living tissues (especially mammalian brain) more transparent for light microscopy, while minimizing or eliminating the toxic side effects of such intervention. A few groundbreaking publications have advanced this field recently, eg. (Ou et al., Science, 2024, Inagaki et al., Nat. Meth. 2026). In this work, the authors apply iodixanol to increase the refractive index (RI) of ACSF, thereby making brain tissues slightly more transparent while minimizing the osmotic stress associated with such intervention.
Strengths:
The approach is methodologically valid, and the authors carefully tested both optical and behavior effects of I-ACSF injection in mice.
Weaknesses:
(1) The authors do not cite a recent key paper in the field of in vivo tissue clearing, against which the results of this work should be compared: Inagaki, S., Nakagawa-Tamagawa, N., Huynh, N.Z. et al. Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo. Nat Methods 23, 839-853 (2026).
(2) Use of iodixanol is not novel for in vivo compatible tuning of refractive index (REF 31, Boothe et al., 2017).
(3) Relatively small increase in RI from ACSF (1.34) to I-ACSF (1.385, by 0.045, slightly above the cytoplasm RI 1.368), especially compared to other intracellular components (lipids and proteins, 1.43-1.55) which are the root causes of light scattering.
(4) As a result, there is very small improvement in tissue transparency (e.g., Figure 1a).
(5) Even at this modest RI increase, mouse behavior differs significantly from controls (Figure 2).
(6) Also, in 2P microscopy experiments (Figure 5), the tissue clearing effect is very modest.
(7) Line 306 calling it a 'dramatic enhancement' is an overstatement.
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