Using Spectral Reflectometry to Determine Myelinated Axon Diameters in a Fixed Mouse Brain Slice

Abstract

Source: Kwon, J., et al., Spectral Reflectometric Microscopy on Myelinated Axons In Situ. J. Vis. Exp. (2018)

This video demonstrates the use of spectral reflectometry with a hyperspectral confocal microscope to analyze interference patterns and determine myelinated axon diameters in fixed brain tissue slices.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Apochromat objective 40×, NA 1.1	Leica Microsystems	15506357	Water-immersion type.
Fluoromyelin Green	Thermo Fisher	F34651
Leica SP8 TCS microscope	Leica Microsystems	SP8
Imaging software	Leica Microsystems	LAS-X
Matlab	MathWorks
Mirror	Thorlabs	PF10-03-P01	Coated with protected silver.
Cover slip	Thermo Fisher	3306	Thickness: #1 (0.13 to 0.17 mm).
Slide glass	Muto Pure Chemicals	5116-20F	Thickness: ~1 mm.
Super glue	Henkel	Loctite 406	Use a dispensing equipment to avoid skin or eye contact.
White-light laser	NKT photonics	EXB-6	EXB-6 was discontinued and replaced by EXU-6.