Quantifying myelin content in brain tissue using color Spatial Light Interference Microscopy (cSLIM).

Matthew J Kuchan,Mikhail E Kandel,Michael Fanous,Laurie A Rund,Catherine Best-Popescu,Gabriel Popescu,Rodney W Johnson,Yuval Garini,Young Jae Lee,Tapas Das,Megan P Caputo

doi:10.1371/journal.pone.0241084

Abstract

Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.

Highlights

Myelin is a critical component of the nervous system white matter
To test the ability of color spatial light interference microscopy (cSLIM) to assess the dry mass content of myelin, we cleared mouse brain sections stained with Luxol Fast Blue (LFB) and imaged the specimens before and after clearing
Since the Luxol Fast Blue stain attaches to the protein content of the myelin, and its hue is still present after clearing (Fig 5D), it is clear that part or all of the lipid content was removed. This confirms that the cSLIM is sensitive to the dry mass of myelin lipids

Summary

Introduction

The myelin sheath surrounds axons and thereby provides the necessary insulation for the efficient transmission of neural electrical signals across brain regions [1]. Myelination of fiber bundles is one of the slowest processes of brain maturation in humans, starting at 16 weeks gestational age and increasing rapidly from 24 weeks through to the perinatal period and continuing until puberty. Quantifying myelin content in brain tissue using color spatial light interference microscopy (cSLIM). Gov/) grant CBET- 0939511 STC, the National Institutes of Health https://www.nih.gov/ grant CA238191, and NRT-UtB 1735252, as well as Abbott Nutrition C3123. Tapas Das and Matthew Kuchan are affiliated with Abbott Nutrition, who provided support in the form of salaries and materials for authors C.B-P. and G.P. but did not have any role in data collection or analysis. The specific roles of these authors are articulated in the ‘author contributions’ section

Methods

Results

Conclusion