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

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.

Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

“…The closeness and extent of overlap for each combination illustrates the minute numerical differences in the images themselves, suggesting that diagnostic capabilities are largely due to the differences in the spatial distributions. This is further substantiated in the statistical differences in dry mass measurements of the same samples found in our previous study 40 only after applying binary myelin masks to the quantitative data.…”

“…Tissues were derived from piglets as described in [16]. Associated diets and gestational sizes, as well as tissue slide preparations, are fully described in 40 . Briefly, piglets were acquired at 2 days of age from the University of Illinois Swine Farm and underwent limited farm processing.…”

“…We have previously analyzed piglet brain tissue using color spatial light interference microscopy (cSLIM) 39,40 , which uses a brightfield objective and an RGB camera, and generates 4 intensity images, one of which is a standard LFB color image. Thus, cSLIM simultaneously yields both a brightfield image and a variations in brain tissue, a strategy undertaken for the first time to our knowledge.…”

Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

“…The closeness and extent of overlap for each combination illustrates the minute numerical differences in the pixels, suggesting that diagnostic capabilities are largely due to differences in spatial distributions and phase density. This is further substantiated in the statistical differences in dry mass measurements of the same samples found in our previous study 40 only after applying binary myelin masks to the quantitative data.…”

“…Associated diets and gestational sizes, as well as tissue slide preparations, are fully described in Ref. 40 . Briefly, piglets were acquired at two days of age from the University of Illinois Swine Farm and underwent limited farm processing.…”