Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy

Abstract

We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.

Figure 1. In vivo imaging of mouse cortex using spectral confocal reflectance microscopy (SCoRe)

(a) Diagram depicting the imaging and optical setup of SCoRe. Three laser wavelengths are emitted simultaneously and reflect off structures in the mouse cortex. Out of focus light is rejected by the pinhole within the microscope, and the reflected light is separated by a prism into three separate photodetectors. (b) Simultaneous imaging of brain vasculature with intravenous injection of fluorescent dextran (red) and combined-wavelength image of reflective fibers (cyan) in the somatosensory cortex. Z-projection over 15 μm. 100 μm scale bar. (c) High magnification images of monochromatic reflective signal captured with the 458, 476 488, 514, 561, and 633 nm lasers and then merged as indicated (bottom panels). 5 μm scale bar. (d–f) Comparison of all lasers to 488, 561 and 633 shows that these three are sufficient for full fiber detection. 5 μm scale bar. (g) Graph showing the reflection intensity along the axon boxed in f demonstrating that lasers of wavelengths 488, 561 and 633 nm have some overlapping but mostly non-overlapping reflection peaks. (h) Graph showing the reflection pattern of similar wavelengths (476 and 488 nm) is mostly overlapping. (i) SCoRe Z-projection (magenta) captured from a Thy1-YFP mouse showing a YFP-labeled axon (green) that is reflective (arrows), however most YFP-labeled axons and all dendrites are not reflective. 50 μm scale bar (experiments were replicated 3 times in n=15 mice).

Figure 2. SCoRe signal is dependent on myelination

(a) In vivo staining of cortical myelin with Fluoromyelin (FM) (red) labels only the reflective fibers (cyan). 10 μm scale bar. (b) In vivo FM staining in the cortex of a Thy1-YFP mouse showing that YFP-labeled axons (green), that are FM negative and therefore unmyelinated axons (arrowhead), are not reflective. Dendrites (arrow), which are never myelinated, are also not reflective. However, all FM positive segments are reflective (cyan). 10 μm scale bar. (c) Example of an axonal bifurcation in a Thy1-YFP mouse imaged in vivo demonstrating that specific parts of an axon that are FM positive are also reflective, however the unmyelinated, FM negative portions of the same axon are not reflective (arrow). 3 μm scale bar. (d) In vivo staining and imaging of myelin in the sciatic nerve with FM (red), reveals the location of nodes of Ranvier (arrows) which lack reflection (cyan). 15 μm scale bar (experiments were replicated 3 times in n=13 mice).

Figure 3. Transcranial time-lapse imaging of the mouse cortex reveals progressive age-dependent myelination

(a) In vivo SCoRe z-projections taken from the mouse somatosensory cortex at various postnatal ages. (b) Images of the same cortical region captured through a thinned skull over four time points showing the appearance of new reflecting fibers. 20 μm scale bars in a–b. (c–e) High magnification z-projections of specific axons that were myelinated between imaging sessions (arrows) at the ages indicated, demonstrating that SCoRe reveals new myelin formation in vivo. (f–h) Z-projections showing repeated imaging of suspected nodes of Ranvier (f) (arrowheads) and stable myelinated axons (g–h) in older animals. 10 μm scale bars in c–h (experiments were replicated 3 times in n=9 mice).

Figure 4. Multicolor reflection spectrum reveals distinct myelin structures in the spinal cord and sciatic nerve in vivo

(a–b) Multicolor SCoRe images captured from the spinal cord (a) and sciatic nerve (b) showing that individual fibers reflect different colors but have a predominant color consistency along each axon. 25 μm scale bars. (c) Two differentially reflecting axons in the sciatic nerve at high-resolution. 10 μm scale bar. (d–f) In vivo SCoRe and fluorescence images captured from an mT/mG mouse expressing tdTomato in myelin sheaths (red) (d) showing Schmidt-Lanterman incisures (arrows) and nodes of Ranvier (arrowheads) (e). Combined reflection image in cyan is shown in composite with tdTomato in (f). 20 μm scale bar in d–f. (g–h) High magnification image of two Schmidt-Lanterman incisures showing SCoRe vertical interference pattern and fluorescent tdTomato (white) overlay (g) and SCoRe alone (h). (i–j) High magnification images of two Schmidt-Lanterman incisures and one node of Ranvier showing all reflected lasers (i) and combined reflection (cyan) with tdTomato fluorescence (red) (j). 4 μm scale bar in g–j (experiments were replicated 3 times in n= 8 mice for spinal cord, n=10 mice for sciatic nerve).

Figure 5. Myelin pathology and human myelinated axons imaged with SCoRe

(a–d) Images captured through a cranial window in P35 wildtype (n=3 mice, 5 replicates) (a–b) and congenitally hypomyelinated shiverer mouse (n=2 mice, 4 replicates) (c–d). In shiverer, we saw small patches of Fluoromyelin (FM)-labeled myelinated axon segments (c), which we never observed in wildtype mice (a). These patches were also reflective only in the region that was FM-labeled (d, arrowheads). 25 μm scale bar in a,c, 5 μm in b,d. (e–f) Images acquired in vivo from the sciatic nerve of an mT/mG mouse with membrane bound tdTomato before (left) and after (right) intraneural injection of the demyelinating agent lysophosphatidylcholine (LPC), showing an acute change in the reflected spectrum (e) and in the tdTomato fluorescence distribution (f). 30 μm scale bar in e–f (n=3 mice, 3 replicates). (g) Photograph showing the setup for SCoRe imaging of the cortex through the pial surface in a fixed human brain explant. (h) Z-projection reflection image obtained from the human brain explant. 30 μm scale bar. (i) High-magnification multicolor (top left) and combined (cyan) images of two myelinated reflective fibers demonstrated by Fluoromyelin labeling (red). 3 μm scale bar.