Coherent Anti-Stokes Raman Spectroscopy to Visualize Myelinated Neurons in Mouse Brain Tissue

Samenvatting

Source: McCullagh, E. A., et al. Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices. J. Vis. Exp. (2022)

The video demonstrates fluorescence and Coherent Anti-Stokes Raman Spectroscopy (CARS) imaging to visualize neuronal architecture in rodent brain tissue. Nissl staining labels cell bodies by binding to ribosomal RNA, while CARS imaging targets lipid-rich myelin through laser-induced signals. Overlaying the fluorescence and CARS images provides a detailed view of cell bodies and myelinated axons.

Protocol

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

1. Staining

1. Stain free floating sections for Nissl, to visualize cell bodies (1:100), in antibody media (AB media: 0.1 M phosphate buffer (PB: 50 mM KH2PO4, 150 mM Na2HPO4), 150 mM NaCl, 3 mM Triton-X, 1% bovine serum albumin (BSA)) for 30 min at room temperature on a standard laboratory shaker.
   1. Protect sections from light using aluminum foil and/or a cover. 550 nm or below wavelengths are compatible with CARS imaging (Figure 1).
      NOTE: While we do not expe....

Resultaten

Figure 1: CARS imaging can be combined with immunofluorescent imaging. The graphs shows that CARS.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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