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.