Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo

Reviewer #1 (Public Review):

Cheng, Yu-Ting, et al. demonstrate the capabilities of three-photon excited fluorescence (3PEF) microscopy for in vivo imaging of the mouse spinal cord. It enables imaging up to ~550 µm in depth, overcoming the limitations of two-photon excited fluorescence (2PEF) microscopy. The authors used 3PEF to visualize and quantify blood flow across different vessel types within the spinal cord and observed the cellular responses following venule occlusion. They showed depth-dependent structural changes in neurites and the behavior of microglia with a high contrast. The findings show that 3PEF can provide high-resolution, multicolor imaging of dynamic cellular interactions and vascular architecture, helping studies of spinal cord physiology and pathology.

The experiments are well done and supported by data but some points need to be clarified:

(1) For the two vs three-photon comparison, the authors should provide more information about how they performed the 2PEF: power and pulse duration. This comparison is primarily focused on imaging depth and signal-to-background ratio (SBR), but imaging speed should also be discussed.

(2) A comparison with state-of-the-art 2PEF would have been more convincing. For instance, the use of adaptive optics, or red-shifted fluorophores allowing better 2PEF SBR, or deeper imaging.

(3) The study focuses on structural imaging and does not provide extensive data on real-time dynamic processes, which could be crucial for understanding rapid cellular responses in the spinal cord.
By addressing these weaknesses, future studies could enhance the applicability and reliability of 3PEF microscopy for spinal cord research.

Reviewer #2 (Public Review):

Summary:

In this work, the authors attempt to advance our capacity to image the intact spinal cord in living mice, with the ultimate goal of allowing optical access to all spinal layers, from the dorsal (sensory-related) to the ventral (motor-related) laminae. They demonstrate the potency of 3-photon excited fluorescence imaging (3PEF) to collect fluorescent signals in anesthetized adult mice to depths of up to 450 µm from the dorsal surface.

Strengths:

• 3PEF is convincingly demonstrated as a significant improvement over previously used 2-photon imaging.

• The images show very good spatial resolution and stable signal-to-noise ratio up to 450 µm from the dorsal surface, providing unprecedented access to intermediate ventral laminae.

Weaknesses:

• The paper in its current form lacks a detailed description of the experimental apparatus used, including its invasiveness (removal of vertebrae and muscles) and its impact on animal behavior. One can hope that, in the future, a similar implantation chamber may be used for awake, freely-moving animals.

• In general, non-optic specialists may find it difficult to appreciate some of the findings due to technical writing at times, and minimally described metrics.

• The possibility that the 3-photon illumination may cause tissue damage, notably by heat induction, is not evaluated or discussed.

• At this stage, no attempt has been made to image cellular activity. The reader should keep in mind that motor neurons, as well as most of their upstream circuits, are located between 500 and 900 µm from the dorsal surface. Hence, although the method is a significant advancement, it still does not allow for the evaluation of morphological (or possibly, activity) changes in the whole spinal cord, particularly excluding motor-related laminae."