Monday, 24th June 2019
    MICROSCOPÍA ÓPTICA Y CONFOCAL
 

Coordinador Científico:
Fco. Javier Díez-Guerra
Responsable Técnico:
Ángeles Muñoz

 

Microscopía Óptica y Confocal

 

SMOC

 

Comunidad de Madrid

 

ÚLTIMAS NOTICIAS

 

INFORMACIÓN Y NOVEDADES - RECOMENDACIONES GENERALES

 

 

ESPAÑOL

Echad un vistazo de vez en cuando a la página de Novedades.

  1. RESERVAS: en la sección Novedades echaros un vistazo a las "Instrucciones y normas para hacer reservas".
  2. IMÁGENES: con mayor antigüedad de dos meses o que no se encuentren en las carpetas previstas para ello serán susceptibles de ser eliminadas sin previo aviso en caso necesario. Nunca guardéis las imágenes en el escritorio. Dejarlas siempre en vuestra carpeta "Imagenes" o "Experiments".
  3. LAMPARA DE FLUORESCENCIA:
    • Antes de encender la lámpara de fluorescencia tocarla. Si está caliente esperar a que se enfríe.
    • Si la persona que viene después os ha dicho que llegará antes de una hora no la apaguéis, ya que es el tiempo mínimo para que se enfríe. Si va a tardar más de ese tiempo mejor apagarla.
  4. LOS LÁSERES DE LOS CONFOCALES conviene encenderlos media hora antes de empezar a usarlos para que alcancen su máxima potencia de excitación.
  5. El microscopio AXIOSKOP-con ccd monocroma también dispone de una cámara ccd de alta sensibilidad para adquirir imágenes digitales.
  6. Miraos la página Software/Adobe photoshop porque encontrareis información útil para procesar vuestras imágenes.
  7. Es recomendable en muchos caso aplicar Filtros de suavizado (Median, Remove haze, etc.) a las imágenes capturadas, sobre todo en Confocales.
  8. Estudios de colocalización y cuantificaciones de fluorescencia. Os recomendamos echar un vistazo a la página dedicada a ello en Protolocos. Cosas que debéis tener en cuenta:
    • Es necesario usar "Background Substraction" y "Shading Correction" en las cámaras ccd
    • Es muy recomendable utilizar la adquisición con "Average" de varias imágenes para mejorar su relación señal-ruido, tanto en microscopios confocales como de campo ancho
    • Es conveniente hacer Deconvolución
    • Sobre todo adquirir las imágenes con la mejor calidad posible, evitando en lo posible la sobreestimación del fondo y la saturación
  9. Para medir distancias en las fotos adquiridas con cámaras ccd tenéis el documento "Parámetros para medir distancias", en las secciones Software/Metamorph y Software/Deconvolución, que os da el valor de micras/pixel que necesitáis para cada objetivo usado. CUIDADO: las medidas son sin usar binning. Con binning el tamaño del pixel aumenta proporcionalemente
  10. Os recomendamos echar un vistazo a la página dedicada a estudios in vivo en el apartado Protocolos.
  11. ¿Qué debo optimizar para obtener buenas inmunofluorescencias?:
    1. El método de fijación (Web "Protocolos/Fijación")
    2. La solución de bloqueo (Web "Protocolos/Inmunofluorescencia" y "Protocolos/Tejidos")
    3. Métodos de eliminación de autofluorescencia (Web "Protocolos/Autofluorescencia")
    4. Incubación con los anticuerpos primario y secundario (Web "Protocolos/Anticuerpos" y "Reactivos y fluoróforos/Sistemas de marcaje")
    5. Diferentes medios de montaje (Web "Reactivos y fluoróforos/Medios de Montaje")
  12. Controles importantes para inmunofluorescencia:
    1. AUTOFLUORESCENCIA, desarrollando el mismo protocolo pero sin anticuerpos primario y secundario
    2. ANTICUERPOS SECUNDARIOS, incubando la muestra sólo con cada uno de los anticuerpos secundarios
    3. CRUCE O INTERFERENCIA DE CANALES, incubando con cada una de las combinaciones de anticuerpo primario y secundario por separado para recoger imágenes en todos los canales con los mismos parámetros de adquisición que los usados en las preparaciones dobles o triples
    4. INCUBACIÓN SIMULTÁNEA FRENTE A LA SECUENCIAL. Aunque la incubación simultánea en un mismo paso con los anticuerpos primarios por un lado y los secundarios por otro es más rápida y cómoda, en algunos casos podría ser preferible incubar secuencialmente primero con uno de los primarios y después con su secundario correspondiente. Así sucesivamente con todos los demás
  13. Uso de proteínas fluorescentes:
    1. Es conveniente que comprobéis los perfiles de excitación y emisión de las proteínas al expresarse en vuestro sistema y condiciones
    2. Descartad problemas de fotoconversión: proteínas fluorescentes que al excitarse en una determinada longitud de onda cambian sus perfiles de emisión y/o excitación, de manera reversible o irreversible
    3. La fijación puede reducir mucho la emisión de fluorescencia. En estos casos puede ser necesario usar anticuerpos específicos
    4. Tenemos una página Web dedicada a las proteínas fluorescentes: Reactivos y fluoróforos/Proteínas fluorescentes

 

ENGLISH

Please, take a look to Novedades from time to time.

  1. BOOKING: you will find help and instructions in "Información y Novedades/Instrucciones y normas para hacer reservas".
  2. IMAGES: older than two months or saved out of user folder in Microscope computers could be deleted if necessary without prior notice. Please, do not save images on Desktop computers. Do it always in "Imagenes" or "Experiments" folders.
  3. FLUORESCENCE LAMPS:
    • Please, touch the lamp before switching it on to test whether it is hot. In that case, wait until it gets cold.
    • The lamp needs around one hour to cool down so if the person behind you is arriving before that time leave it ON.
  4. CONFOCAL LASERS should be turned on at least half an hour before use to get their maximum power of excitation.
  5. The AXIOSKOP-con ccd monocroma microscope has also got a high-sensitivity ccd camera to acquire digital images.
  6. You will find useful information for image processing in the Software/Adobe photoshop SMOC's Web section.
  7. The use of image FILTERS is highly recommended (Median, Remove haze, etc.) after acquisition, specially in confocal images.
  8. COLOCALIZATION AND QUANTIFICATION STUDIES. We have some recommendations and useful information in our Web page section "Protolocos":
    • It is important to apply "Background Substraction" and "Shading Correction" with ccd cameras
    • Acquisiton with "Average" improves the signal to noise ratio of confocal and widefield images
    • Deconvolution is also very usefull to improve image quality
    • In general, it is very important to get the best image quality in terms of signal-to-noise ratio, avoiding background and saturation
  9. The document "Parámetros para medir distancias", in the Web sections Software/Metamorph and Software/Deconvolución, is neccesary to MEASURE DISTANCES IN IMAGES ACQUIRED WITH CCD CAMERAS. In this document you can get the value “microns/pixel” needed for each objective and microscope. WARNING: values indicated have been got without any binning. Pixel size increases with binning.
  10. We also recommend taking a look to our Web section PROCOLOS/ESTUDIOS "IN VIVO".
  11. ¿Where can I get some advices to get GOOD IMMUNOFLUORESCENCES?:
    1. Fixation methods (Web section "Protocolos/Fijación")
    2. Blocking solutions (Web sections "Protocolos/Inmunofluorescencia" and "Protocolos/Tejidos")
    3. Autofluorescence removal methods (Web section "Protocolos/Autofluorescencia")
    4. Primary and secondary antibody incubation (Web sections "Protocolos/Anticuerpos" and "Reactivos y fluoróforos/Sistemas de marcaje")
    5. Mounting methods (Web section "Reactivos y fluoróforos/Medios de Montaje")
  12. CONTROLS for immunofluorescence:
    1. AUTOFLUORESCENCE: develop every protocol step but without primary and secondary antibodies
    2. SECONDARY ANTIBODIES: incubation with just one of the secondary antibodies per sample
    3. BLEEDTHROUGH/CROSSTALK: incubation with just one of the combination of primary and secondary antibodies per sample, to acquire images with the same settings used for multiple antibodies samples
    4. SIMULTANEOUS OR SEQUENTIAL INCUBATION. Although incubation with all primary antibodies simultaneously as a first step and with all secondary ones in a second step can be faster, sequential incubation might be recommended in some cases: in this case, the sample is incubated with one of the primary antibody first and then with its corresponding secondary one, and so on
  13. FLUORESCENT PROTEINS (FPs):
    1. Check FPs excitation and emission profiles expressed in your system and conditions
    2. Rule out photoconversion problems: in some circumstances FP emission profile can change when exciting at some wavelengths. You should check it to avoid artifacts
    3. Fixation can reduce their fluorescence and lifetime. The use of primary antibodies against FPs is recommended in this case
    4. You will find additional information in our Web section "Reactivos y fluoróforos/Proteínas fluorescentes"

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