Name: coherent anti-stokes raman spectroscopy (cars) application for imaging myelination in brain slices
Uploaded: 2022-07-22T14:00:00
Description: Watch this Scientific Journal Video about Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices at JoVE.com

Supported by NIH R01 DC 17924, R01 DC 18401 (Klug), and NIH 1R15HD105231-01, T32DC012280, and FRAXA (McCullagh). The CARS imaging was performed in the Advanced Light Microscopy Core part of the NeuroTechnology Center at the University of Colorado Anschutz Medical Campus supported in part by NIH P30 NS048154 and NIH P30 DK116073.

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
<ol>
	<li>Use C57BL/6J (stock #000664) mice (Mus musculus) obtained from The Jackson Laboratory or Mongolian gerbils (Meriones unguiculatus) originally obtained from Charles River.</li>
</ol>
2. Tissue preparation</p...

<ol>
	<li>Cole, K., Curtis, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+impedance+of+the+squid+giant+axon+during+activity.">Electric impedance of the squid giant axon during activity.</a> The Journal of General Physiology. 22 (5), 649-670 (1939).</li><li>Cole, K. S., Curtis, H. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+properties+and+design+principles+of+axons.">Integrative properties and design principles of axons.</a> International Review of Neurobiology. 18, 1-40 (1975).</li><li>Fitzhugh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computation+of+impulse+initiation+and+saltatory+conduction+in+a+myelinated+nerve+fiber.">Computation of impulse initiation and saltatory conduction in a myelinated nerve fiber.</a> Biophysical Journal. 2 (1), 11-21 (1962).</li><li>Zalc, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acquisition+of+myelin:+a+success+story.">The acquisition of myelin: a success story.</a> Novartis Foundation Symposium. 276, 275-281 (2006).</li><li>Salzer, J. L., Zalc, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination.">Myelination.</a> Current Biology. 26 (20), 971-975 (2016).</li><li>Boullerne, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+myelin.">The history of myelin.</a> Experimental Neurology. 283, 431-445 (2016).</li><li>Kuhn, S., Gritti, L., Crooks, D., Dombrowski, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocytes+in+development,+myelin+generation+and+beyond.">Oligodendrocytes in development, myelin generation and beyond.</a> Cells. 8 (11), 1424 (2019).</li><li>Saab, A. S., Nave, K. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+dynamics:+protecting+and+shaping+neuronal+functions.">Myelin dynamics: protecting and shaping neuronal functions.</a> Current Opinion in Neurobiology. 47, 104-112 (2017).</li><li>Chomiak, T., Hu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+optimal+value+of+the+g-Ratio+for+myelinated+fibers+in+the+rat+CNS?+A+theoretical+approach.">What is the optimal value of the g-Ratio for myelinated fibers in the rat CNS? A theoretical approach.</a> PLOS ONE. 4 (11), 7754 (2009).</li><li>Ford, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+Ranvier+node+and+internode+properties+in+myelinated+axons+to+adjust+action+potential+timing.">Tuning of Ranvier node and internode properties in myelinated axons to adjust action potential timing.</a> Nature Communications. 6, 8073 (2015).</li><li>Stange-Marten, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input+timing+for+spatial+processing+is+precisely+tuned+via+constant+synaptic+delays+and+myelination+patterns+in+the+auditory+brainstem.">Input timing for spatial processing is precisely tuned via constant synaptic delays and myelination patterns in the auditory brainstem.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (24), 4851-4858 (2017).</li><li>Bu, J., Banki, A., Wu, Q., Nishiyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+NG2++glial+cell+proliferation+and+oligodendrocyte+generation+in+the+hypomyelinating+mutant+shiverer.">Increased NG2+ glial cell proliferation and oligodendrocyte generation in the hypomyelinating mutant shiverer.</a> Glia. 48 (1), 51-63 (2004).</li><li>Pacey, L. K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+myelination+in+a+mouse+model+of+fragile+X+syndrome.">Delayed myelination in a mouse model of fragile X syndrome.</a> Human Molecular Genetics. 22 (19), 3920-3930 (2013).</li><li>Green, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clemastine+fumarate+as+a+remyelinating+therapy+for+multiple+sclerosis+(ReBUILD):+a+randomised,+controlled,+double-blind,+crossover+trial.">Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled, double-blind, crossover trial.</a> Lancet. 390 (10111), 2481-2489 (2017).</li><li>Jeon, S. J., Ryu, J. H., Bahn, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+translational+control+of+fragile+X+mental+retardation+protein+on+myelin+proteins+in+neuropsychiatric+disorders.">Altered translational control of fragile X mental retardation protein on myelin proteins in neuropsychiatric disorders.</a> Biomolecules &amp; Therapeutics. 25 (3), 231-238 (2017).</li><li>Barak, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+deletion+of+Gtf2i,+associated+with+Williams+syndrome,+causes+behavioral+and+myelin+alterations+rescuable+by+a+remyelinating+drug.">Neuronal deletion of Gtf2i, associated with Williams syndrome, causes behavioral and myelin alterations rescuable by a remyelinating drug.</a> Nature Neuroscience. 22 (5), 700-708 (2019).</li><li>Phan, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myelin-related+transcriptomic+profile+is+shared+by+Pitt-Hopkins+syndrome+models+and+human+autism+spectrum+disorder.">A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndrome models and human autism spectrum disorder.</a> Nature Neuroscience. 23 (3), 375-385 (2020).</li><li>Lucas, A., Poleg, S., Klug, A., McCullagh, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+deficits+in+the+auditory+brainstem+of+a+mouse+model+of+fragile+X+syndrome.">Myelination deficits in the auditory brainstem of a mouse model of fragile X syndrome.</a> Frontiers in Neuroscience. 15, 1536 (2021).</li><li>Wang, H., Fu, Y., Zickmund, P., Shi, R., Cheng, J. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coherent+anti-stokes+raman+scattering+imaging+of+axonal+myelin+in+live+spinal+ttissues.">Coherent anti-stokes raman scattering imaging of axonal myelin in live spinal ttissues.</a> Biophysical Journal. 89 (1), 581-591 (2005).</li><li>Kim, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+coherent+anti-stokes+raman+spectroscopy+images+intact+atheromatous+lesions+and+concomitantly+identifies+distinct+chemical+profiles+of+atherosclerotic+lipids.">Multiplex coherent anti-stokes raman spectroscopy images intact atheromatous lesions and concomitantly identifies distinct chemical profiles of atherosclerotic lipids.</a> Circulation Research. 106 (8), 1332-1341 (2010).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Tu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-floating+Immunostaining+of+Mouse+Brains.">Free-floating Immunostaining of Mouse Brains.</a> Journal of Visualized Experiments. (176), e62876 (2021).</li><li>. Fluorescence SpectraViewer Available from: <a target="_blank" href="https://www.thermofisher.com/order/fluorescence-spectraviewer">https://www.thermofisher.com/order/fluorescence-spectraviewer</a> (2022)</li><li>Held, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+centrale+geh&#246;rleitung.">Die centrale geh&#246;rleitung.</a> Arch Anat Physiol Anat Abt. 17, 201-248 (1893).</li><li>Sherman, D. L., Brophy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axon+ensheathment+and+myelin+growth.">Mechanisms of axon ensheathment and myelin growth.</a> Nature Reviews Neuroscience. 6 (9), 683-690 (2005).</li><li>Gruchot, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+remyelination+failure+in+multiple+sclerosis.">The molecular basis for remyelination failure in multiple sclerosis.</a> Cells. 8 (8), 825 (2019).</li><li>Rivera, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+pathway+in+the+age-related+decline+of+oligodendrocyte+regeneration.">Epidermal growth factor pathway in the age-related decline of oligodendrocyte regeneration.</a> Frontiers in Cellular Neuroscience. 16, 838007 (2022).</li><li>K&#250;tna, V., O'Leary, V. B., Hoschl, C., Ovsepian, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+demyelination+and+neurodegeneration+associated+with+mTORC1+hyperactivity+may+contribute+to+the+developmental+onset+of+autism-like+neurobehavioral+phenotype+in+a+rat+model.">Cerebellar demyelination and neurodegeneration associated with mTORC1 hyperactivity may contribute to the developmental onset of autism-like neurobehavioral phenotype in a rat model.</a> Autism Research: Official Journal of the International Society for Autism Research. 15 (5), 791-805 (2022).</li><li>Ozsv&#225;r, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+lipid+debris+accumulation+caused+by+cuprizone+induced+myelin+degradation+in+different+CNS+areas.">Quantitative analysis of lipid debris accumulation caused by cuprizone induced myelin degradation in different CNS areas.</a> Brain Research Bulletin. 137, 277-284 (2018).</li><li>Prineas, J. 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A growing body of literature emphasizes the role of myelin in brain function13,16,21,28. Moreover, we know that myelination thickness and myelination pattern can change in several neurological conditions such as multiple sclerosis (reviewed in29), aging (reviewed in30), autism20,31, and ma...

Action potentials are the basic unit of information in the brain, and action potential propagation through axons forms one pillar of information processing1,2,3. Neurons typically receive afferent inputs from multiple other neurons and integrate these inputs within a given narrow time window4,5. 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 regenerated repeatedly along the axon to ensure reliable propagation6. In most neurons of jawed vertebrates (gnathostomes) axons are surrounded by a sheath of myelin, which is a lipid-rich substance produced by nearby oligodendrocytes or Schwann cells, which are types of glial cells (reviewed in7,8). This myelin sheath electrically insulates the axon, reducing its capacitance and allowing action potential propagation efficiently, quickly, and with lower energy consumption. Myelin does not cover the axon uniformly, but it sheaths the axon in segments that have short gaps in between them, called the nodes of Ranvier (reviewed in9,10). Both myelination thickness, which controls the level of electrical insulation of an axon, and the spacing of the nodes of Ranvier, which control the frequency with which action potentials are regenerated along an axon, influence the speed of action potential propagation (reviewed in11).
There is a large body of literature suggesting that myelination thickness affects the speed of action potential propagation in axons12,13,14. Moreover, alterations in axon myelination can result in a number of CNS deficits15,16,17,18,19,20,21. It is therefore not surprising that the focus of many research efforts involves the measurement and characterization of axon myelination. Measurements of myelin thickness have most commonly been done with electron microscopy, a technique that requires a significant amount of tissue preparation and is challenging to use in combination with immunohistochemistry. However, there is also a faster and simpler technique to measure axon myelination that is based on Coherent Anti-Stokes Raman Spectroscopy (CARS). A CARS laser can be tuned to various frequencies and when tuned to frequencies that are suitable to excite lipids, myelin can be imaged without the need for any additional labels22. The lipid imaging can be combined with standard immunohistochemistry such that lipids can be imaged together with several fluorescent channels23. Imaging myelination with CARS is significantly faster than electron microscopy and has a resolution that is, albeit lower than EM, sufficient to detect even small differences in myelination in the same type of axons.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthetic:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL disposable syringe with needle 27 GA x 0.5&#34;</td>
			<td>Exel int</td>
			<td>260040</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatal +</td>
			<td>Vortech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 8mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard tweezers</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Fisher Chemical</td>
			<td>SF994 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14063-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Kelly hemostats</td>
			<td>Fine Science Tools</td>
			<td>13019-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore H2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle tip, 23 GA x 1&#34;</td>
			<td>BD precision glide</td>
			<td>305193</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobase</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>pump with variable flow or equivalent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Chemical</td>
			<td>s271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodiumphosphate dibasic</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL vial with 4% PFA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bochem Chemical Spoon 180mm</td>
			<td>Bochem</td>
			<td>230331000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14063-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15011-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pair of fine (Graefe) tweezers</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Shallow glass or plastic tray, approximately 10&#34; x 10&#34;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard tweezers</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Blunt</td>
			<td>Fine Science Tools</td>
			<td>14000-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicing:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, plant</td>
			<td>RPI</td>
			<td>9002-18-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000s</td>
			<td></td>
		</tr>
		<tr>
			<td>well plate</td>
			<td>Alkali Sci.</td>
			<td>TPN1048-NT</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AB Media:</td>
			<td></td>
			<td></td>
			<td>1n 1,000 mL of Millipore H2O</td>
		</tr>
		<tr>
			<td>Phosphate buffered (PB):</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobase</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosohate Dibasic</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (Bovine serum albumin)</td>
			<td>Sigma life science</td>
			<td>A2153-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Chemical</td>
			<td>s271-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma - Aldrich</td>
			<td>x100-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Nissl 435/455</td>
			<td>Invitrogen</td>
			<td>N21479</td>
			<td></td>
		</tr>
		<tr>
			<td>CARS:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APE&#160;picoemerald laser</td>
			<td>Angewandte Physik &#38; Elektronik&#160;GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter (420-520 nm)</td>
			<td>Chroma Technology</td>
			<td>HQ470/100m-2P</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter (500-530 nm)</td>
			<td>Chroma Technology</td>
			<td>HQ515/30m-2P</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filters (640-680 nm)</td>
			<td>Chroma Technology</td>
			<td>HQ660/40m-2P</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cut Transfer pipet</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic longpass 565 nm</td>
			<td>Chroma Technology</td>
			<td>565dcxr</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic longpass 585 nm</td>
			<td>Chroma Technology</td>
			<td>585dcxr</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic shortpass 750 nm</td>
			<td>Chroma Technology</td>
			<td>T750spxrxt</td>
			<td></td>
		</tr>
		<tr>
			<td>glass bottom culture dish</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>glass weight (10 mm x 10 mm boro rod)</td>
			<td>Allen Scientific Glass Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>multiphoton shortpass emission filter 680 nm</td>
			<td>Chroma Technology</td>
			<td>ET680sp-2p8</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

coherent anti-stokes raman spectroscopy (cars) application for imaging myelination in brain slices

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.

One of the biggest advantages of CARS microscopy over other techniques is the compatibility with fluorescent imaging23. Figure 1 shows the CARS spectra compared to Nissl tagged with immunofluorescent marker showing little/no overlap in spectra. Figure 2 illustrates the laser set up for CARS in combination with confocal microscopy. Figure 3 demonstrates two representative images, one as a single stack and one ...

Watch this Scientific Journal Video about Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices at JoVE.com

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

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.

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

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...

This technique can image myelin in brain tissue sections. myelination plays an important role in the conduction of action potentials and understanding myelin will help us understand brain function. The main advantage of the CARS technique over, for example, electron microscopy is that CARS is a light microscopy technique, and can easily be combined with other fluorescence imaging, such as used in immunohistochemistry.Several medical conditions are due to alterations in myelination, for example, multiple sclerosis, aging, and autism. Understanding these alterations will help us understand the underlying medical conditions. In this case, the CARS laser is tuned to image lipids or, specifically, CH2 bonds.In the brain, the largest source of lipids is myelin. Thus, this is a technique to image myelin in brain tissue. It should be possible to use the same method for other types of tissue when imaging lipids is of interest.Tuning and aligning the CARS laser requires significant expertise, and is best performed by a laser or light microscopy expert. After preparing the tissues as described in the manuscript, stain free-floating sections for nisel and antibody media to visualize cell bodies, then incubate the sections on a standard laboratory shaker for 30 minutes at room temperature. Before bringing the samples to the microscope, turn on and warm up the CARS laser for at least one hour, then align the CARS laser by spatially overlapping the pump and stokes laser beams, and adjust the delay between the two laser beams.Color the condenser optics and the diaphragm of the microscope for forward CARS imaging, then adjust the external periscope to center the spatially overlapped two lasers onto the scanning head mirrors of the microscope. For best forward CARS non-descanned detection. make sure the condenser is color lured.For immunofluorescence, confocal imaging, and cars imaging, fit the CARS laser with both forward and epi CARS non-descanned detectors by incorporating a confocal microscope equipped with visible lasers for fluorescence imaging. Now, place the sections in a culture dish with a cover slip bottom, and PBS to avoid drying the tissue. Also, use a glass weight to keep the tissue near the cover slip.Using the graphic user interface, set the image acquisition parameters for cars and missile fluorescence confocal imaging. Place the sample on the microscope stage, focus the sample, and capture the images. CARS'spectra range from 640 to 660 nanometers and there appears to be no overlap in spectra with nisel-tagged immunofluorescent marker, indicating that CARS signals can be used with immunofluorescent signals.Nisel was used to visualize the cell bodies of Mongolian gerbil and mouse brains, and CARS'imaging was used to visualize the myelin sheath, indicating that this technique could be used across species. Both sets of images show a section of the medial nucleus of the trapezoid body in the brain stem. The resolution of CARS is lower than with electron microscope and is on the order of several hundred nanometers.The images can be analyzed with standard image analysis software, such as ImageJ to quantify the parameters of interest, for example, myelin thickness or length.

Research

JoVE Journal

Neuroscience

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...