Este trabajo fue financiado por el Instituto de ciencias básicas (IBS-R015-D1) y programa de investigación de ciencia básica a través de la nacional investigación Fundación de Corea (NRF) financiado por el Ministerio de Educación (2017R1A6A1A03015642).

Todos los procedimientos quirúrgicos fueron aprobados por el institucional Animal Care y el Comité uso (IACUC) de la Universidad de Sungkyunkwan.
1. preparación de la muestra
Nota: Autoclave todos los instrumentos quirúrgicos antes de manejo de animales. Llevar a cabo todos actuación quirúrgica en una sala dedicada a procedimientos quirúrgicos. Guantes y batas quirúrgicas estériles deben llevarse por todo el personal en la sala quirúrgica en todo momento.
<ol><li>
Fijación del tejido
	<ol>
<li>Preparar dos jeringas de 10 mL, que cada uno relleno con solución salina tamponada con fosfato (PBS) y 4% paraformaldehido (PFA) en PBS.</li>
		<li>Anestesiar un ratón de 7-12 semanas de edad, (C57BL/6J o las líneas de ratón con ambos sexos) mediante la administración de una mezcla de zoletil y xilacina (1:1, 20 mg/kg cada uno) o un régimen de anestesia conveniente alternativa (por ejemplo, mezcla de 80 mg/kg ketamina y 10 mg/kg xilacina) con una inyección intraperitoneal.</li>
		<li>Una vez que el ratón ha alcanzado un plano quirúrgico de anestesia (uso el método de respuesta a la pizca del dedo del pie), exponer el corazón cortando la piel y la caja torácica con las tijeras.</li>
		<li>Cortar la aurícula derecha con pequeñas tijeras para drenar la sangre y la perfusión de la solución PBS y PFA secuencialmente a través del ventrículo izquierdo con una aguja (tasa de perfusión = 4 mL/min, volumen = 10 mL de cada solución). Nota: El ratón se convierte en duro si el procedimiento se ha completado con éxito. El uso de ungüento oftálmico o esterilización no es necesario ya que el procedimiento es terminal. El procedimiento de fijación puede ser saltado. Sin embargo, este paso se recomienda para reducir las deformaciones estructurales incluyendo daño a los tejidos mecánicos e hinchazón axonal isquémico. Para más detalles sobre la fijación de tejido, consulte el artículo de Gage, G. J. et al. 10
</li>
	</ol>
</li>
	<li>
Preparación de la rebanada del cerebro
	<ol>
<li>Decapitar el ratón con tijeras quirúrgicas a través del tórax ventral y el abdomen.</li>
		<li>Quitar el cuero cabelludo y el periostio con una tijeras convenientemente pequeña hasta exponer completamente el cráneo.</li>
		<li>Romper el hueso frontal y cortar el cráneo a lo largo de la sutura sagital del hueso occipital con pequeñas tijeras con cuidado de no para dañar el cerebro.</li>
		<li>Retire suavemente el cráneo y la duramadre secuencialmente con unas pinzas finas.</li>
		<li>Extraer el cerebro con una cuchara de plástico suave, teniendo cuidado de no para dañar el tejido.</li>
		<li>Enjuagar y remojar el cerebro extraído en una solución PFA 4% durante 30 min a 4 ° C.</li>
		<li>Cortar el cerebro fijado en 100 – 150 μm secciones con un vibratome. Nota: Para más detalles sobre la preparación del tejido, consulte el artículo de Segev et al. 11. para los procedimientos posteriores, el tejido debe ser cortado en secciones más fino que el espesor del espaciador.</li>
	</ol>
</li>
	<li>
Montaje de la rebanada del cerebro
	<ol>
<li>Preparar 1 diapositiva de cristal y 2 vasos cuadrados de cubierta (22 × 22 × 0,17 mm) para cada rebanada de tejido.</li>
		<li>Cortar uno de los vidrios de cubierta cuadrados en la mitad (dos piezas rectangulares) usando un cortador de vidrio.</li>
		<li>Conecte los dos de los vidrios de cubierta a la mitad utilizando un pegamento en el cristal de la diapositiva. Nota: El espacio entre los dos vasos a la mitad debe ser ligeramente mayor que el tamaño de la rodaja de tejido.</li>
		<li>Cosecha de la rebanada del cerebro utilizando un cepillo o una pipeta y coloque el tejido en el cristal deslizante entre el espaciador. Tenga cuidado de no para doblar el tejido.</li>
		<li>Dispensar 100 μL de PBS en la superficie del tejido.</li>
		<li>Lugar el otros cuadrada cubierta de vidrio en la parte superior del tejido. Evitar la inclusión de burbujas de aire durante esta etapa.</li>
		<li>Selle alrededor del vidrio de cubierta con un esmalte de uñas (o adhesivos como alternativa adecuados) para evitar la evaporación de PBS y la contaminación por el polvo durante la sesión de imágenes posteriores. Nota: El experimentador puede detenerse en esta etapa.</li>
	</ol>
</li>
</ol>2. calibración
<ol><li>Encienda el microscopio al menos 1 h antes de la proyección de imagen para permitir la estabilización térmica del láser (véase Tabla de materiales) de la fuente. A continuación, abra el software de análisis espectral (véase Tabla de materiales) y haga clic en el botón adquirir GUI (figura 3a).
</li>
	<li>Abra el software de disparo para el láser de luz blanca (WLL) y tubo fotomultiplicador (PMT) (figura 3a).</li>
	<li>Seleccione el modo xyΛ en la lista desplegable en el Modo de adquisición, entonces, el modo de entrada de láser automáticamente cambia de porcentaje constante a Potencia constante. Desactive la casilla de verificación de Movimiento automático de SP. Y a continuación, establezca la ventana espectral del láser de entrada 470-670 nm y el tamaño de paso espectral a 4 nm en Λ-excitación Lambda Scan configuración (figura 3b).</li>
	<li>ESTABLECER el rango espectral del PMT a 450 690 doble clic o mover la barra de ajuste para la gama espectral (Figura 3C). Nota: Este espectro debe incluir el ancho de banda completo de la fuente de entrada.</li>
	<li>Seleccione un objetivo de inmersión en agua, convenientemente con una alta apertura numérica (NA > 0,7) y dar cualquier valor a poder PMT y láser para activar el botón de configuración AOBS y Live Scan . Cambiar la trayectoria óptica control de reflejo en la Configuración AOBS (Figura 3d).</li>
	<li>Montar un espejo de referencia (véase Tabla de materiales) en la platina del microscopio. La superficie del espejo debe encararse contra la lente del objetivo. Si no es fácil de poner el espejo en la platina del microscopio, enlazar un espejo sobre la placa plana (e.g. cristal de la diapositiva).</li>
	<li>Control de la platina del microscopio para alinear el plano focal a la superficie del espejo.</li>
	<li>Ajustar la ganancia de la PMT y la potencia del láser teniendo en cuenta el rango dinámico de los detectores y luego cambiar el modo de entrada de láser por ciento constante a Potencia constante. Nota: Como es típico, un PMT ganancia de 500 (V) y una potencia de láser relativa de 0,1% utilizan a 570 nm.</li>
	<li>Confirmar en modo pseudo-color para comprobar que no hay ninguna saturación en toda la gama de longitud de onda. Si se observa saturación, bajar la potencia del láser (figura 3a).</li>
	<li>Ejecutar la adquisición Lambda Scan .</li>
	<li>Quite el espejo de la etapa y repetir la misma adquisición sin una muestra para obtener la referencia (es decir, offset oscuro).</li>
	<li>Guardar los datos en un formato TIF Multistacked .</li>
</ol>3. adquisición de la imagen SpeRe
<ol><li>Coloque el tejido montado en la platina del microscopio. Áspero alinear el tejido para el plano focal de la lente del objetivo, utilice el modo de campo amplio de la fluorescencia a través de un ocular.</li>
	<li>Con la Live Scan en control de la platina del microscopio para alinear el plano focal para la región de interés en el tejido. Para evitar el ruido de fondo de la hoja de cubierta, seleccione la región de destino de al menos 15 de μm de profundidad desde la interfaz de tejido de vidrio.</li>
	<li>Adquirir la pila de imagen espectral para la región de destino utilizando el mismo procedimiento como se describe en los pasos 2.1-2.10.</li>
	<li>Guardar los datos para el tejido y el oscuro offset en formato TIF Multistacked . Nota: El experimentador puede detenerse en esta etapa.</li>
</ol>4. procesamiento y análisis de imágenes
<ol></ol>5. línea base corrección y análisis de señales SpeRe
<ol><li>Reste los espectros de desplazamiento de los espectros del espejo de referencia y los tejidos del cerebro.</li>
	<li>Normalizar cada espectro dividiendo la intensidad máxima del espectro.</li>
	<li>Dividir el espectro normalizado del axón por el espectro normalizado del espejo de referencia y restar el offset de DC del espectro normalizado.</li>
	<li>Medir la frecuencia de wavenumber ajustando el espectro adquirido a una sinusoidal (véase Tabla de materiales) de la curva y luego convertir el número adquirido a periodicidad por tomando recíproco.</li>
	<li>Convertir la periodicidad proporción al diámetro del axón mediante la ecuación en la figura 4C.</li>
</ol>

<ol>
	<li>Fernandez-Moran, H., Finean, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+and+low-angle+x-ray+diffraction+studies+of+the+nerve+myelin+sheath.">Electron microscope and low-angle x-ray diffraction studies of the nerve myelin sheath.</a> Journal of Cell Biology. 3, (1957).</li><li>Blaurock, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spaces+between+membrane+bilayers+within+PNS+myelin+as+characterized+by+X-ray+diffraction.">The spaces between membrane bilayers within PNS myelin as characterized by X-ray diffraction.</a> Brain Research. 210, 383-387 (1981).</li><li>Shim, 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=Super-resolution+fluorescence+imaging+of+organelles+in+live+cells+with+photoswitchable+membrane+probes.">Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes.</a> Proceedings of the National Academy of Sciences. , 13978-13983 (2012).</li><li>Urban, N. T., Willig, K. I., Hell, S. W., N&#228;gerl, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+nanoscopy+of+actin+dynamics+in+synapses+deep+inside+living+brain+slices.">STED nanoscopy of actin dynamics in synapses deep inside living brain slices.</a> Biophysics Journal. 101, 1277-1284 (2011).</li><li>Manley, S., 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=High-density+mapping+of+single-molecule+trajectories+with+photoactivated+localization+microscopy.">High-density mapping of single-molecule trajectories with photoactivated localization microscopy.</a> Nature Methods. 5, 155-157 (2008).</li><li>Peters, A., Sethares, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+remyelination+during+aging+of+the+primate+central+nervous+system?.">Is there remyelination during aging of the primate central nervous system?.</a> Journal of Comparative Neurology. 460, 238-254 (2003).</li><li>De Campos Vidal, B., Silveira Mello, M. L., Caseiro-Filho, A. C., Godo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+properties+of+the+myelin+sheath.">Anisotropic properties of the myelin sheath.</a> Acta Histochemica. 66, 32-39 (1980).</li><li>Schain, A. J., Hill, R. A., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+in+vivo+imaging+of+myelinated+axons+in+health+and+disease+with+spectral+confocal+reflectance+microscopy.">Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy.</a> Nature Medicine. 20, 443-449 (2014).</li><li>Kwon, 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=Label-free+nanoscale+optical+metrology+on+myelinated+axons+in+vivo.">Label-free nanoscale optical metrology on myelinated axons in vivo.</a> Nature Communication. 8, 1832 (2017).</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> JoVE. (65), e3564 (2012).</li><li>Segev, A., Garcia-Oscos, F., Kourrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+Patch-clamp+Recordings+in+Brain+Slices.">Whole-cell Patch-clamp Recordings in Brain Slices.</a> JoVE. (112), e54024 (2016).</li><li>Waldchen, S., Lehmann, J., Klein, T., Van De Linde, S., Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+cell+damage+in+live-cell+super-resolution+microscopy.">Light-induced cell damage in live-cell super-resolution microscopy.</a> Scientific Reports. 5, 15348 (2015).</li><li>Chang, J. 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=Iterative+expansion+microscopy.">Iterative expansion microscopy.</a> Nature Methods. 14, 593-599 (2017).</li><li>Polishchuk, R. S., 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=Correlative+light-electron+microscopy+reveals+the+tubular-saccular+ultrastructure+of+carriers+operating+between+Golgi+apparatus+and+plasma+membrane.">Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.</a> Journal of Cell Biology. 148, 45-58 (2000).</li></ol>

Los autores declaran que compiten intereses financieros: Kwon J. y M. Choi son inventores de la tecnología pendiente de patente se describe en este artículo.

SpeRe es una modalidad de etiqueta-libre nuevo basada en interferometría espectral, que por primera vez, ofrece la información de nanoescala en axones mielinizados energizados. En el actual protocolo de adquisición, la resolución espacial para el diámetro del axon es del orden de 10 nm. Por otra parte, SpeRe utiliza órdenes de la magnitud dosis más baja de luz en comparación con otras Microscopías de súper-resolución; por lo tanto, es libre de fototoxicidad y photobleaching. SpeRe proporcionaría una nueva ví...

En el mamífero sistema nervioso, mielina proporciona integridad axonal y conducción nerviosa rápida enwrapping las fibras de axones con varias capas envolturas membranosas. Su estructura multicapa se compone de alterna de nanoescala películas delgadas compuestas de membranas del plasma (~ 5 nm), citosol (~ 3 nm) y espacios extracelulares (~ 7 nm)1,2. Microscopía óptica, incluyendo la microscopía de superresolución recientes, no son adecuados para la observación de la dinámica de la mielina de escala nanométrica debido a su insuficiente resolución debido a la difracción óptica3,4,5. Aunque la microscopía electrónica puede proporcionar detalles finos de la nanoestructura de la mielina, no es compatible con los sistemas biológicos vivos debido a preparaciones de muestra altamente invasiva que involucra la fijación química y ultrasectioning6,7 . Hasta hace poco, no se ha producido ninguna técnica aplicable para observar dinámicas de nanoescala de axones myelinated en situ.
Schain Ciencias et al había divulgado previamente que axones myelinated exhiben colores reflectancia luz8. Adoptando el análisis espectroscópico de la luz reflejada, hemos ideado una nueva modalidad de imagen para imágenes a nanoescala de axones myelinated, llamado reflectometría espectral (SpeRe)9. SpeRe se basa en la interferencia de película delgada que ocurre en la estructura de varias capas de la vaina de mielina (figura 1). Por simulación óptica en varios axones, ha revelado que el espectro de reflectancia es una función periódica del número y su periodicidad (<img alt="Equation 1" src="/files/ftp_upload/57965/57965eq1.jpg">) es inversamente proporcional al diámetro del axón (d). Esta relación simple (<img alt="Equation 2" src="/files/ftp_upload/57965/57965eq2.jpg">) ofrece fácil cuantificación de diámetro de axón de los datos de SpeRe. Utilizando esto, nos reveló el frecuente axón abultamiento bajo craneoencefálico leve en nuestro informe anterior.
El sistema SpeRe está basado en la microscopia confocal y consiste en una fuente de láser especializado y filtros (figura 2). La fuente de entrada es un láser de luz blanca, proporciona banda ancha salida espectral visible a infrarrojo regiones. Para el análisis espectral, el sistema está equipado con dos dispositivos de acusto-óptica: un filtro sintonizable de acusto-óptica (AOTF) para la entrega de una longitud de onda seleccionada de la fuente de entrada de banda ancha y un divisor de viga de acusto-óptico (AOBS) para orientar el seleccionado refleja longitud de onda del detector. El software para microscopía confocal hiperespectral (véase Tabla de materiales) ofrece una opción de análisis espectral personalizable para adquirir secuencialmente las imágenes de reflectividad en distintas longitudes de onda en la entrada. Además, la aberración cromática críticamente puede interferir en la medición espectral; por lo tanto, se recomienda el uso de una lente de objetivo de apochromat.
De nota, láseres de luz blanca producen una salida espectral desigual y los componentes ópticos también afectan el perfil espectral. Por lo tanto, los espectros adquiridos deben ser calibrados para el posterior análisis cuantitativo. Un espejo de plata protegido se utiliza normalmente como una referencia, que proporciona una reflexión casi constante (> 97%) en la región visible completo. Luego se dividen los espectros adquiridos por los espectros de referencia desde el espejo.
El tamaño de paso espectral para el análisis espectral determina la velocidad de adquisición; por lo tanto, debe ser optimizado. Como un axon más grande tiene un mayor periodo espectral, requiere más espectral muestreo. Por ejemplo, un axón con un diámetro de 10 μm, uno de los más grandes axones fisiológicos, tiene un periodo espectral de 8 nm. Mediante la aplicación de criterios de muestreo de Nyquist, se empleó el intervalo de muestreo espectral de 4 nm para cubrir todos los axones fisiológicos en los tejidos nerviosos de ratón. Este enfoque por lo general toma varios segundos para un análisis espectral completo y así no es adecuado para aplicaciones en vivo , donde el movimiento fisiológico (por ejemplo, la respiración y del latido del corazón) interfiere adquisición espectral estable. Ya resolvimos esta cuestión por equipar un microscopio vertical personalizado, diseñado para adquirir la gama completa para cada punto usando un espectrómetro de matriz (adquisición velocidad ≈ 30 m por píxel).
En este informe, describimos un protocolo detallado en la proyección de imagen de SpeRe en una rebanada del cerebro fija, que puede realizarse en un microscopio hiperespectral comercial (véase Tabla de materiales). Así, el protocolo puede ser completado por experimentadores sin conocimientos en instrumentación óptica. También cubrimos los posibles problemas y solución de problemas para la adquisición y análisis de datos SpeRe.

<table>
	<tbody>
		<tr>
			<td>Glass cutter</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>-</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>Apochromat objective 40&#215;, NA 1.1</td>
			<td>Leica Microsystems</td>
			<td>15506357</td>
			<td>Water-immersion type</td>
		</tr>
		<tr>
			<td>Fluoromyelin Green</td>
			<td>Thermo Fisher</td>
			<td>F34651</td>
			<td>Alternatively, Fluoromyelin Red (F34652) can be used.</td>
		</tr>
		<tr>
			<td>Leica SP8 TCS microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8</td>
			<td>Refer to the &#34;Configuration of microscope&#34; in Introduction Section for details.</td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Leica Microsystems</td>
			<td>LAS-X</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Thorlabs</td>
			<td>PF10-03-P01</td>
			<td>Coated with protected silver.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Life technologies</td>
			<td>14190-136</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosolution</td>
			<td>BP031a</td>
			<td>4% v/v in PBS</td>
		</tr>
		<tr>
			<td>Cover slip</td>
			<td>Thermo Fisher</td>
			<td>3306</td>
			<td>Thickness: #1 (0.13 to 0.17 mm)</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td>Muto Pure Chemicals</td>
			<td>5116-20F</td>
			<td>Thickness: ~1 mm</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel</td>
			<td>Loctite 406</td>
			<td>Use a dispensing equipment to avoid skin or eye contact.</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Brainetree Scientific</td>
			<td>BS-8000 DUAL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1200S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>White-light laser</td>
			<td>NKT photonics</td>
			<td>EXB-6</td>
			<td>EXB-6 was discontinued and replaced by EXU-6.</td>
		</tr>
	</tbody>
</table>

spectral reflectometric microscopy on myelinated axons in situ

En un mamífero sistema nervioso, mielina proporciona un aislamiento eléctrico enwrapping las fibras del axón en una espiral de varias capas. Inspirado por su arquitectura subcelular altamente organizado, recientemente desarrollamos una nueva modalidad de imágenes, llamada reflectometría espectral (SpeRe), que permite la proyección de imagen de nanoescala etiqueta-libre sin precedentes de los axones myelinated vivo in situ. El principio subyacente es obtener información nano-estructural analizando el espectro de reflectancia de la estructura subcelular de varias capas. En este artículo se describe un protocolo detallado paso a paso para realizar una básica SpeRe la proyección de imagen de los tejidos nerviosos mediante un sistema microscopio confocal comercial, equipado con un láser de luz blanca y un filtro sintonizable. Cubrimos los procedimientos de preparación de muestras, adquisición de datos espectrales y procesamiento de imagen para obtener información de nano-estructural.

Según el protocolo, un trozo de cerebro fija fue preparado con coloración exógena, un fluoróforo dirigidos a la mielina (véase Tabla de materiales). SpeRe la proyección de imagen se realizó en el segmento del cerebro con un microscopio confocal comercial hiperespectral en conjunto (figura 4a) la proyección de imagen confocal de la fluorescencia. SpeRe, entrada intensidad óptica fue establecido como 5 μW/μm2 con un tiempo...

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Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Microscopía espectral reflectométrico en axones mielinizados en Situ

Aquí, presentamos un protocolo paso a paso de axones myelinated en un trozo de cerebro fija usando una nanoescala libre etiqueta técnica basado en reflectometría spectral de la proyección de imagen de la proyección de imagen.

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

spectral-reflectometric-microscopy-on-myelinated-axons-in-situ

Research

JoVE Journal

Neuroscience

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

7.3K vistas.  Sungkyunkwan University. Aquí, presentamos un protocolo paso a paso de axones myelinated en un trozo de cerebro fija usando una nanoescala libre etiqueta técnica basado en reflectometría spectral de la proyección de imagen de la proyección de imagen.

Video: Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Visualización en vivo de las vesículas sinápticas En Drosophila segmentaria axones larvas

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Investigación

Neurociencias

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

Imágenes en vivo de los axones de la raíz dorsal después de rizotomía

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

Imagen confocal espectral de marcado con fluorescencia receptores nicotínicos en Knock-en ratones con administración crónica de nicotina

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagen Funcional Utilizando ultrasonidos hematoencefálica perturbación de la barrera y la RM-manganeso mayor

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

Analizar el desarrollo de células murinas Schwann largo de los axones en crecimiento

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo de dos fotones de imágenes de dependientes Experience-Los cambios moleculares en las neuronas corticales

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Laser Nanocirugía del cerebelo Los axones<em&gt; En Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Imágenes en vivo de nicotina señalización de calcio inducido y neurotransmisor lanzamiento Junto ventrales hipocampo axones

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

Rápido<em&gt; In Situ</em&gt; La hibridación usando sondas de oligonucleótidos en Cerebro paraformaldehído-prefijo de ratas con síndrome de serotonina

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

Determinación de los fotorreceptores de la célula de sensibilidad espectral en un modelo de insectos<em&gt; En Vivo</em&gt; registros intracelulares

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...