ultrathin sectioning for epon post-embedding correlative light and electron microscopy (clem)

Nuevo protocolo para la conservación simultánea de fluorescencia y ultraestructura

Fluorescence microscopy (FM) can be used to obtain the localization and distribution of the target protein. However, the context that surrounds the target protein is lost, which is crucial for investigating the target protein thoroughly. Electron microscopy (EM) has the highest imaging resolution, providing several subcellular details. Nevertheless, EM lacks target labeling. By accurately merging the fluorescence image taken by FM with the gray image acquired by EM, correlative light and electron microscopy (CLEM) can combine the information obtained by these two imaging modes1,2,3,4.
CLEM is a trade-off between fluorescence and the ultrastructure1. Because of the limitations of current fluorescent proteins and the traditional EM sample preparation procedures, especially the use of osmic acid (OsO4) and hydrophobic resins such as Epon, the ultrastructure always compromises fluorescence5. OsO4 is an indispensable reagent in EM sample preparation, which is used to improve the contrast of EM images. Compared with other embedding resins, Epon is superior in ultrastructure preservation and sectioning properties5. However, no fluorescent proteins can retain the fluorescence signal after the treatment of OsO4 and Epon embedding6. To overcome the limitations of fluorescent proteins, pre embedding CLEM was developed, in which FM imaging is done before EM sample preparation6. However, the drawback of pre embedding CLEM is the imprecise registration between the FM and the EM images5.
On the contrary, post embedding CLEM method performs the FM imaging after the EM sample preparation, the registration accuracy of which can reach 6-7 nm5,6. To retain the fluorescence of fluorescent proteins, very low concentrations of OsO4 (0.001%)3 or the high-pressure frozen (HPF) and freeze substitution (FS) EM preparation methods4,7 have been used at the expense of compromised ultrastructure or the contrast of the EM image. The development of mEos4b greatly promotes the progress of post embedding CLEM, although glycidyl methacrylate is used as the embedding resin5. With the development of mEosEM, which can survive the OsO4 staining and Epon embedding, Epon post embedding super-resolution CLEM was achieved for the first time, maintaining the fluorescence and ultrastructure simultaneously6. After mEosEM, several fluorescent proteins that can survive the OsO4 staining and the Epon embedding were developed8,9,10,11. This greatly promotes the development of CLEM.
There are three key aspects to Epon post-embedding CLEM. The first is the fluorescent protein, which should maintain the fluorescent signal after EM sample preparation. According to our experience, mScarlet is superior to other reported fluorescent proteins. The second is how to find the same cell imaged by FM imaging in EM imaging. To solve this problem, we improve the procedure for this step so that one can readily find the targeted cell. The last is the method to align the FM image with the EM image. Here, we detail the registration between the FM and the EM images. In this protocol, we express mScarlet in VGLUT2 neurons and demonstrate that mScarlet can target secondary lysosomes using Epon post-embedding CLEM. We provide step-by-step details for Epon post-embedding CLEM, without compromising the fluorescence and the ultrastructure.

Autor destacado: Avances en microscopía electrónica y de luz correlativa con preservación de proteínas fluorescentes

Microscopía electrónica y de luz correlativa de inclusión posterior a Epon

Correlative light and electron microscopy is a completely stable microscopy technique, which can combine the localization information provided by fluorescent microscopy and is the ultra structure by electron microscopy. However, the mentality of this technique is to preserve the fluorescence of after you have a simple preparation. Using this fluorescent protein, we can achieve correlative light and the electron microscope which can maintain fluorescence and ultra structure simultaneously.

Corte ultrafino para microscopía correlativa de luz y electrónica posterior a la inclusión de epon (CLEM)

Para empezar, toma un bloque de tejido cerebral de ratón y recorta su superficie en un trapecio en ángulo recto. Usando un cuchillo de diamante con un ultra micrótomo, corte secciones con un grosor de 100 nanómetros. Separe una sola sección de la banda de sección y déjela flotar en el baño de agua.Agregue una gota de agua destilada doble al centro de la cubierta de vidrio recubierta de nanopartículas de oro. Use un bucle de muestra para tomar una sección e invertirla en la superficie de la gota de agua. Retire el bucle de muestra dejando la sección en la superficie de la gota de agua.Luego, con un rotulador, dibuja un anillo alrededor de la sección en el lado opuesto de la cubierta de vidrio.

ultrathin-sectioning-for-epon-post-embedding-correlative-light

Research

JoVE Journal

Biology

Ultrathin Sectioning for Epon Post-Embedding Correlative Light and Electron Microscopy (CLEM)

88 vistas.  Fujian Medical University. Para empezar, toma un bloque de tejido cerebral de ratón y recorta su superficie en un trapecio en ángulo recto. Usando un cuchillo de diamante con un ultra micrótomo, corte secciones con un grosor de 100 nanómetros. Separe una sola sección de la banda de sección y déjela flotar en el baño de agua.Agregue una gota de agua destilada doble al centro de la cubierta de vidrio recubierta de nanopartículas de oro. Use un bucle de muestra para tomar una sección e invertirla en la su...

Video: Corte ultrafino para microscopía correlativa de luz y electrónica posterior a la inclusión de epon (CLEM)

Epon Post Embedding Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.

We present a detailed protocol for Epon post-embedding correlative light and electron microscopy using a fluorescent protein called mScarlet. This method can maintain the fluorescence and the ultrastructure simultaneously. This technique is amenable to a wide variety of biological applications.

Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence ...

Investigación

Biología

Creating the Navigation Map for CLEM and Preparation of the Ultrathin Sections for Electron Microscopy

Creación del mapa de navegación para CLEM y preparación de las secciones ultrafinas para microscopía electrónica 

Image Analysis of Brightfield and Fluorescence Images of the Mouse Brain Sections for Epon Post-Embedding Correlative Light and Electron Microscopy

Análisis de imágenes de campo claro e imágenes de fluorescencia de las secciones cerebrales de ratón para microscopía electrónica y de luz correlativa posterior a la inclusión de Epon