Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Summary

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Abstract

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Introduction

Action potentials are the basic unit of information in the brain, and action potential propagation through axons forms one pillar of information processing^1,2,3. Neurons typically receive afferent inputs from multiple other neurons and integrate these inputs within a given narrow time window^4,5. Therefore, the mechanisms of action potential propagation in axons have received a significant amount of attention from investigators.

When propagating through an axon, an action potential is regene....

Protocol

All experiments complied with all applicable laws, National Institutes of Health guidelines, and were approved by the University of Colorado Anschutz Institutional Animal Care and Use Committee.

1. Animals

1. Use C57BL/6J (stock #000664) mice (Mus musculus) obtained from The Jackson Laboratory or Mongolian gerbils (Meriones unguiculatus) originally obtained from Charles River.

2. Tissue preparation

Discussion

A growing body of literature emphasizes the role of myelin in brain function^13,16,21,28. Moreover, we know that myelination thickness and myelination pattern can change in several neurological conditions such as multiple sclerosis (reviewed in²⁹), aging (reviewed in³⁰), autism^20,31, and ma.......

Materials

Name	Company	Catalog Number	Comments
Anesthetic:
1 mL disposable syringe with needle 27 GA x 0.5"	Exel int	260040
Fatal +	Vortech
Surgery:
Spring Scissors - 8mm Cutting Edge	Fine Science Tools	15024-10
Standard tweezers	Fine Science Tools	11027-12
Perfusion:
4% Paraformaldehyde	Fisher Chemical	SF994 (CS)
Fine Scissors - Sharp	Fine Science Tools	14063-11
Kelly hemostats	Fine Science Tools	13019-14
Millipore H2O
Needle tip, 23 GA x 1"	BD precision glide	305193
Phosphate buffered saline (PBS):
Potassium chloride	Sigma	P9333
Potassium phosphate monobase	Sigma	P5655
pump with variable flow or equivalent
Sodium chloride	Fisher Chemical	s271-1
Sodiumphosphate dibasic	Sigma	S7907
Dissection:
50 mL vial with 4% PFA
Bochem Chemical Spoon 180mm	Bochem	230331000
Fine Scissors - Sharp	Fine Science Tools	14063-11
Noyes Spring Scissors	Fine Science Tools	15011-12
Pair of fine (Graefe) tweezers	Fine Science Tools	11050-10
Shallow glass or plastic tray, approximately 10" x 10"
Standard tweezers	Fine Science Tools	11027-12
Surgical Scissors - Blunt	Fine Science Tools	14000-20
Slicing:
Agar, plant	RPI	9002-18-0
Vibratome	Leica	VT1000s
well plate	Alkali Sci.	TPN1048-NT
Staining:
AB Media:			1n 1,000 mL of Millipore H2O
Phosphate buffered (PB):
Potassium Phosphate Monobase	Sigma	P5655
Sodium Phosohate Dibasic	Sigma	S7907
BSA (Bovine serum albumin)	Sigma life science	A2153-100g
Sodium Chloride	Fisher Chemical	s271-1
Triton X-100	Sigma - Aldrich	x100-500ml
Nissl 435/455	Invitrogen	N21479
CARS:
APE picoemerald laser	Angewandte Physik & Elektronik GmbH
bandpass filter (420-520 nm)	Chroma Technology	HQ470/100m-2P
bandpass filter (500-530 nm)	Chroma Technology	HQ515/30m-2P
bandpass filters (640-680 nm)	Chroma Technology	HQ660/40m-2P
Confocal microscope	Olympus	FV1000
Cut Transfer pipet	Fisher	13-711-7M
dichroic longpass 565 nm	Chroma Technology	565dcxr
dichroic longpass 585 nm	Chroma Technology	585dcxr
dichroic shortpass 750 nm	Chroma Technology	T750spxrxt
glass bottom culture dish	MatTek	P35G-0-10-C
glass weight (10 mm x 10 mm boro rod)	Allen Scientific Glass Inc
multiphoton shortpass emission filter 680 nm	Chroma Technology	ET680sp-2p8
PBS